Glycosylation of the N-terminal potential N-glycosylation sites in the human α1,3-fucosyltransferase V and -VI (hFucTV and -VI)

DEFF Research Database (Denmark)

Christensen, Lise Lotte; Bross, Peter Gerd; Ørntoft, Torben Falck

2000-01-01

Human alpha1,3-fucosyltransferase V and -VI (hFucTV and -VI) each contain four potential N-glycosylation sites (hFucTV: Asn60, Asn105, Asn167 and Asn198 and hFucTVI: Asn46, Asn91, Asn153 and Asn184). Glycosylation of the two N-terminal potential N-glycosylation sites (hFucTV: Asn60, Asn105 and h......FucTVI: Asn46 and Asn91) have never been studied in detail. In the present study, we have analysed the glycosylation of these potential N-glycosylation sites. Initially, we compared the molecular mass of hFucTV and -VI expressed in COS-7 cells treated with tunicamycin with the mass of the proteins...... in untreated cells. The difference in molecular mass between the proteins in treated and untreated cells corresponded to the presence of at least three N-linked glycans. We then made a series of mutants, in which the asparagine residues in the N-terminal potential N-glycosylation sites were replaced...

Unusual glycosylation of proteins: Beyond the universal sequon and other amino acids.

Science.gov (United States)

Dutta, Devawati; Mandal, Chhabinath; Mandal, Chitra

2017-12-01

Glycosylation of proteins is the most common, multifaceted co- and post-translational modification responsible for many biological processes and cellular functions. Significant alterations and aberrations of these processes are related to various pathological conditions, and often turn out to be disease biomarkers. Conventional N-glycosylation occurs through the recognition of the consensus sequon, asparagine (Asn)-X-serine (Ser)/threonine (Thr), where X is any amino acid except for proline, with N-acetylglucosamine (GlcNAc) as the first glycosidic linkage. Usually, O-glycosylation adds a glycan to the hydroxyl group of Ser or Thr beginning with N-acetylgalactosamine (GalNAc). Protein glycosylation is further governed by additional diversifications in sequon and structure, which are yet to be fully explored. This review mainly focuses on the occurrence of N-glycosylation in non-consensus motifs, where Ser/Thr at the +2 position is substituted by other amino acids. Additionally, N-glycosylation is also observed in other amide/amine group-containing amino acids. Similarly, O-glycosylation occurs at hydroxyl group-containing amino acids other than serine/threonine. The neighbouring amino acids and local structural features around the potential glycosylation site also play a significant role in determining the extent of glycosylation. All of these phenomena that yield glycosylation at the atypical sites are reported in a variety of biological systems, including different pathological conditions. Therefore, the discovery of more novel sequence patterns for N- and O-glycosylation may help in understanding the functions of complex biological processes and cellular functions. Taken together, all these information provided in this review would be helpful for the biological readers. Copyright © 2017 Elsevier B.V. All rights reserved.

Engineer medium and feed for modulating N-glycosylation of recombinant protein production in CHO cell culture

DEFF Research Database (Denmark)

Fan, Yuzhou; Kildegaard, Helene Faustrup; Andersen, Mikael Rørdam

2017-01-01

Chinese hamster ovary (CHO) cells have become the primary expression system for the production of complex recombinant proteins due to their long-term success in industrial scale production and generating appropriate protein N-glycans similar to that of humans. Control and optimization of protein N......-glycosylation is crucial, as the structure of N-glycans can largely influence both biological and physicochemical properties of recombinant proteins. Protein N-glycosylation in CHO cell culture can be controlled and tuned by engineering medium, feed, culture process, as well as genetic elements of the cell...

[Conformation analysis of the N-glycosylation site Asn-X-Thr/Ser in glycoproteins].

Science.gov (United States)

Avanov, A Ia; Lipkind, G M

1990-03-01

Theoretical conformational analysis of oligopeptides CH3CO-Asn-X-Thr-NHCH3 (X = Gly, Ala, Pro), modelling N-glycosylation site, and their glycosylated derivatives CH3CO-(GlcNAc beta 1-4GlcNAc beta 1) Asn-X-Thr-NHCH3 has been carried out. Active conformations of the site are found, corresponding to structural prerequisities of N-glycosylation: Asn residue's position in beta-turn and hydrogen bond formation between side chains of Asn and Thr/Ser residues. In this case the L conformation of the central residue X is most probable. Since Pro residue does not possess this conformation, sequences with X = Pro are not glycosylated. It is shown that glycosylation of the above-mentioned sites is accompanied by reorientation of the Asn residue's side chains.

Functional Role of N-Linked Glycosylation in Pseudorabies Virus Glycoprotein gH.

Science.gov (United States)

Vallbracht, Melina; Rehwaldt, Sascha; Klupp, Barbara G; Mettenleiter, Thomas C; Fuchs, Walter

2018-05-01

Many viral envelope proteins are modified by asparagine (N)-linked glycosylation, which can influence their structure, physicochemical properties, intracellular transport, and function. Here, we systematically analyzed the functional relevance of N-linked glycans in the alphaherpesvirus pseudorabies virus (PrV) glycoprotein H (gH), which is an essential component of the conserved core herpesvirus fusion machinery. Upon gD-mediated receptor binding, the heterodimeric complex of gH and gL activates gB to mediate fusion of the viral envelope with the host cell membrane for viral entry. gH contains five potential N-linked glycosylation sites at positions 77, 162, 542, 604, and 627, which were inactivated by conservative mutations (asparagine to glutamine) singly or in combination. The mutated proteins were tested for correct expression and fusion activity. Additionally, the mutated gH genes were inserted into the PrV genome for analysis of function during virus infection. Our results demonstrate that all five sites are glycosylated. Inactivation of the PrV-specific N77 or the conserved N627 resulted in significantly reduced in vitro fusion activity, delayed penetration kinetics, and smaller virus plaques. Moreover, substitution of N627 greatly affected transport of gH in transfected cells, resulting in endoplasmic reticulum (ER) retention and reduced surface expression. In contrast, mutation of N604, which is conserved in the Varicellovirus genus, resulted in enhanced in vitro fusion activity and viral cell-to-cell spread. These results demonstrate a role of the N-glycans in proper localization and function of PrV gH. However, even simultaneous inactivation of all five N-glycosylation sites of gH did not severely inhibit formation of infectious virus particles. IMPORTANCE Herpesvirus infection requires fusion of the viral envelope with cellular membranes, which involves the conserved fusion machinery consisting of gB and the heterodimeric gH/gL complex. The bona fide

In-depth mapping of the mouse brain N-glycoproteome reveals widespread N-glycosylation of diverse brain proteins.

Science.gov (United States)

Fang, Pan; Wang, Xin-Jian; Xue, Yu; Liu, Ming-Qi; Zeng, Wen-Feng; Zhang, Yang; Zhang, Lei; Gao, Xing; Yan, Guo-Quan; Yao, Jun; Shen, Hua-Li; Yang, Peng-Yuan

2016-06-21

N-glycosylation is one of the most prominent and abundant posttranslational modifications of proteins. It is estimated that over 50% of mammalian proteins undergo glycosylation. However, the analysis of N-glycoproteins has been limited by the available analytical technology. In this study, we comprehensively mapped the N-glycosylation sites in the mouse brain proteome by combining complementary methods, which included seven protease treatments, four enrichment techniques and two fractionation strategies. Altogether, 13492 N-glycopeptides containing 8386 N-glycosylation sites on 3982 proteins were identified. After evaluating the performance of the above methods, we proposed a simple and efficient workflow for large-scale N-glycosylation site mapping. The optimized workflow yielded 80% of the initially identified N-glycosylation sites with considerably less effort. Analysis of the identified N-glycoproteins revealed that many of the mouse brain proteins are N-glycosylated, including those proteins in critical pathways for nervous system development and neurological disease. Additionally, several important biomarkers of various diseases were found to be N-glycosylated. These data confirm that N-glycosylation is important in both physiological and pathological processes in the brain, and provide useful details about numerous N-glycosylation sites in brain proteins.

Genes Involved in the Endoplasmic Reticulum N-Glycosylation Pathway of the Red Microalga Porphyridium sp.: A Bioinformatic Study

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Oshrat Levy-Ontman

2014-02-01

Full Text Available N-glycosylation is one of the most important post-translational modifications that influence protein polymorphism, including protein structures and their functions. Although this important biological process has been extensively studied in mammals, only limited knowledge exists regarding glycosylation in algae. The current research is focused on the red microalga Porphyridium sp., which is a potentially valuable source for various applications, such as skin therapy, food, and pharmaceuticals. The enzymes involved in the biosynthesis and processing of N-glycans remain undefined in this species, and the mechanism(s of their genetic regulation is completely unknown. In this study, we describe our pioneering attempt to understand the endoplasmic reticulum N-Glycosylation pathway in Porphyridium sp., using a bioinformatic approach. Homology searches, based on sequence similarities with genes encoding proteins involved in the ER N-glycosylation pathway (including their conserved parts were conducted using the TBLASTN function on the algae DNA scaffold contigs database. This approach led to the identification of 24 encoded-genes implicated with the ER N-glycosylation pathway in Porphyridium sp. Homologs were found for almost all known N-glycosylation protein sequences in the ER pathway of Porphyridium sp.; thus, suggesting that the ER-pathway is conserved; as it is in other organisms (animals, plants, yeasts, etc..

N-glycosylation by N-acetylglucosaminyltransferase V enhances the interaction of CD147/basigin with integrin β1 and promotes HCC metastasis.

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Cui, Jian; Huang, Wan; Wu, Bo; Jin, Jin; Jing, Lin; Shi, Wen-Pu; Liu, Zhen-Yu; Yuan, Lin; Luo, Dan; Li, Ling; Chen, Zhi-Nan; Jiang, Jian-Li

2018-05-01

While the importance of protein N-glycosylation in cancer cell migration is well appreciated, the precise mechanisms by which N-acetylglucosaminyltransferase V (GnT-V) regulates cancer processes remain largely unknown. In the current study, we report that GnT-V-mediated N-glycosylation of CD147/basigin, a tumor-associated glycoprotein that carries β1,6-N-acetylglucosamine (β1,6-GlcNAc) glycans, is upregulated during TGF-β1-induced epithelial-to-mesenchymal transition (EMT), which correlates with tumor metastasis in patients with hepatocellular carcinoma (HCC). Interruption of β1,6-GlcNAc glycan modification of CD147/basigin decreased matrix metalloproteinase (MMP) expression in HCC cell lines and affected the interaction of CD147/basigin with integrin β1. These results reveal that β1,6-branched glycans modulate the biological function of CD147/basigin in HCC metastasis. Moreover, we showed that the PI3K/Akt pathway regulates GnT-V expression and that inhibition of GnT-V-mediated N-glycosylation suppressed PI3K signaling. In summary, β1,6-branched N-glycosylation affects the biological function of CD147/basigin and these findings provide a novel approach for the development of therapeutic strategies targeting metastasis. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Viral Restriction Activity of Feline BST2 Is Independent of Its N-Glycosylation and Induction of NF-κB Activation.

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Weiran Wang

Full Text Available BST2 (CD317, tetherin, HM1.24 is an interferon-inducible transmembrane protein which can directly inhibit the release of enveloped virus particles from infected cells, and its anti-viral activity is reported to be related to the specific topological arrangement of its four structural domains. The N-terminal cytoplasmic tail of feline BST2 (fBST2 is characterized by a shorter N-terminal region compared to those of other known homologs. In this study, we investigated the functional impact of modifying the cytoplasmic tail region of fBST2 and its molecular mechanism. The fBST2 protein with the addition of a peptide at the N-terminus retained anti-release activity against human immunodeficiency virus type-1 and pseudovirus based on feline immunodeficiency virus at a weaker level compared with the wild-type fBST2. However, the fBST2 protein with addition of a peptide internally in the ectodomain proximal to the GPI anchor still retained its anti-viral activity well. Notably, the N-glycosylation state and the cell surface level of the N-terminally modified variants were unlike those of the wild-type protein, while no difference was observed in their intracellular localizations. However, in contrast to human BST2, the wild-type fBST2 did not show the ability to activate NF-κB. Consistent with previous reports, our findings showed that adding a peptide in the cytoplasmic tail region of fBST2 may influence its anti-viral activity. The shorter N-terminal cytoplasmic region of fBST2 compared with human BST2 did not apparently affect its anti-viral activity, which is independent of its N-glycosylation and ability to activate NF-κB.

Revealing the mechanisms of protein disorder and N-glycosylation in CD44-hyaluronan binding using molecular simulation

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Olgun eGuvench

2015-06-01

Full Text Available The extracellular N-terminal hyaluronan binding domain (HABD of CD44 is a small globular domain that confers hyaluronan (HA binding functionality to this large transmembrane glycoprotein. When recombinantly expressed by itself, HABD exists as a globular water-soluble protein that retains the capacity to bind HA. This has enabled atomic-resolution structural biology experiments that have revealed the structure of HABD and its binding mode with oligomeric HA. Such experiments have also pointed to an order-to-disorder transition in HABD that is associated with HA binding. However, it had remained unclear how this structural transition was involved in binding since it occurs in a region of HABD distant from the HA-binding site. Furthermore, HABD is known to be N-glycosylated, and such glycosylation can diminish HA binding when the associated N-glycans are capped with sialic acid residues. The intrinsic flexibility of disordered proteins and of N-glycans makes it difficult to apply experimental structural biology approaches to probe the molecular mechanisms of how the order-to-disorder transition and N-glycosylation can modulate HA binding by HABD. We review recent results from molecular dynamics simulations that provide atomic-resolution mechanistic understanding of such modulation to help bridge gaps between existing experimental binding and structural biology data. Findings from these simulations include: Tyr42 may function as a molecular switch that converts the HA binding site from a low affinity to a high affinity state; in the partially-disordered form of HABD, basic amino acids in the C-terminal region can gain sufficient mobility to form direct contacts with bound HA to further stabilize binding; and terminal sialic acids on covalently-attached N-glycans can form charge-paired hydrogen bonding interactions with basic amino acids that could otherwise bind to HA, thereby blocking HA binding to glycosylated CD44 HABD.

Distribution of N-glycosylation sequons in proteins: how apart are they?

DEFF Research Database (Denmark)

Rao, Shyama Prasad; Buus, Ole Thomsen; Wollenweber, Bernd

2011-01-01

of experimentally confirmed eukaryotic N-glycoproteins we analyzed the relative position and distribution of sequons. N-Glycosylation probability was found to be lower in the termini of protein sequences compared to the mid region. N-glycosylated sequons were found much farther from C terminus compared to the N......N-glycosylation is a common protein modification process, which affects a number of properties of proteins. Little is known about the distribution of N-glycosylation sequons, for example, the distance between glycosylated sites and their position in the protein primary sequence. Using a large set......-terminus of the protein sequence and this effect was more pronounced for NXS sequons. The distribution of sequons, modeled based on balls-in-boxes classical occupancy, showed a near-maximum probability. Considerable proportion of sequons was found within a distance of ten amino acids, indicating that the steric hindrance...

Glycosylation in HIV-1 envelope glycoprotein and its biological implications

KAUST Repository

Ho, Yung Shwen; Saksena, Nitin K.

2013-01-01

architecture, also controls intra- and inter-clade genetic variations. Discerning intra- and inter-clade glycosylation variations could therefore yield important information for understanding the molecular and biological differences between HIV clades and may

N-linked glycosylation of the immunoglobulin variable region

NARCIS (Netherlands)

van de Bovenkamp, Fleur S.; Derksen, Ninotska I. L.; Ooijevaar-de Heer, Pleuni; van Schie, Karin A.; Kruithof, Simone; Berkowska, Magdalena A.; van der Schoot, C. Ellen; Ijspeert, Hanna; van der Burg, Mirjam; Gils, Ann; Hafkenscheid, Lise; Toes, René E. M.; Rombouts, Yoann; Plomp, Rosina; Wuhrer, Manfred; van Ham, S. Marieke; Vidarsson, Gestur; Rispens, Theo

2018-01-01

N-glycosylation sites are introduced at positions in which glycans can affect antigen binding as a result of a specific clustering of progenitor glycosylation sites in the germline sequences of variable domain genes. By analyzing multiple human monoclonal and polyclonal (auto)antibody responses, we

Mapping N-linked Glycosylation Sites in the Secretome and Whole Cells of Aspergillus niger Using Hydrazide Chemistry and Mass Spectrometry

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Wang, Lu; Aryal, Uma K.; Dai, Ziyu; Mason, Alisa C.; Monroe, Matthew E.; Tian, Zhixin; Zhou, Jianying; Su, Dian; Weitz, Karl K.; Liu, Tao; Camp, David G.; Smith, Richard D.; Baker, Scott E.; Qian, Weijun

2012-01-01

Protein glycosylation is known to play an essential role in both cellular functions and the secretory pathways; however, little information is available on the dynamics of glycosylated N-linked glycosites of fungi. Herein we present the first extensive mapping of glycosylated N-linked glycosites in industrial strain Aspergillus niger by applying an optimized solid phase enrichment of glycopeptide protocol using hydrazide modified magnetic beads. The enrichment protocol was initially optimized using mouse plasma and A. niger secretome samples, which was then applied to profile N-linked glycosites from both the secretome and whole cell lysates of A. niger. A total of 847 unique N-linked glycosites and 330 N-linked glycoproteins were confidently identified by LC-MS/MS. Based on gene ontology analysis, the identified N-linked glycoproteins in the whole cell lysate were primarily localized in the plasma membrane, endoplasmic reticulum, golgi apparatus, lysosome, and storage vacuoles. The identified N-linked glycoproteins are involved in a wide range of biological processes including gene regulation and signal transduction, protein folding and assembly, protein modification and carbohydrate metabolism. The extensive coverage of glycosylated N-linked glycosites along with identification of partial N-linked glycosylation in those enzymes involving in different biochemical pathways provide useful information for functional studies of N-linked glycosylation and their biotechnological applications in A. niger.

Biochemical Importance of Glycosylation of Plasminogen Activator Inhibitor-1

DEFF Research Database (Denmark)

Gils, Ann; Pedersen, Katrine Egelund; Skottrup, Peter Durand

2003-01-01

The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential target for anti-thrombotic and anti-cancer therapy. PAI-1 has 3 potential sites for N-linked glycosylation. We demonstrate here that PAI-1 expressed recombinantly or naturally by human cell lines display a heterogeneous glycosyla...

Glycosylation Engineering

DEFF Research Database (Denmark)

Clausen, Henrik; Wandall, Hans H.; Steentoft, Catharina

2017-01-01

Knowledge of the cellular pathways of glycosylation across phylogeny provides opportunities for designing glycans via genetic engineering in a wide variety of cell types including bacteria, fungi, plant cells, and mammalian cells. The commercial demand for glycosylation engineering is broad......, including production of biological therapeutics with defined glycosylation (Chapter 57). This chapter describes how knowledge of glycan structures and their metabolism (Parts I–III of this book) has led to the current state of glycosylation engineering in different cell types. Perspectives for rapid...

Stability of Curcuma longa rhizome lectin: Role of N-linked glycosylation.

Science.gov (United States)

Biswas, Himadri; Chattopadhyaya, Rajagopal

2016-04-01

Curcuma longa rhizome lectin, a mannose-binding protein of non-seed portions of turmeric, is known to have antifungal, antibacterial and α-glucosidase inhibitory activities. We studied the role of complex-type glycans attached to asparagine (Asn) 66 and Asn 110 to elucidate the role of carbohydrates in lectin activity and stability. Apart from the native lectin, the characteristics of a deglycosylated Escherichia coli expressed lectin, high-mannose oligosaccharides at both asparagines and its glycosylation mutants N66Q and N110Q expressed in Pichia pastoris, were compared to understand the relationship between glycosylation and activity. Far UV circular dichroism (CD) spectra, fluorescence emission maximum, hemagglutination assay show no change in secondary or tertiary structures or sugar-binding properties between wild-type and aforementioned recombinant lectins under physiological pH. But reduced agglutination activity and loss of tertiary structure are observed in the acidic pH range for the deglycosylated and the N110Q protein. In thermal and guanidine hydrochloride (GdnCl)-induced unfolding, the wild-type and high-mannose lectins possess higher stability compared with the deglycosylated recombinant lectin and both mutants, as measured by a higher Tm of denaturation or a greater free energy change, respectively. Reversibility experiments after thermal denaturation reveal that deglycosylated proteins tend to aggregate during thermal inactivation but the wild type shows a much greater recovery to the native state upon refolding. These results suggest that N-glycosylation in turmeric lectin is important for the maintenance of its proper folding upon changes in pH, and that the oligosaccharides help in maintaining the active conformation and prevent aggregation in unfolded or partially folded molecules. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Functional Divergence in the Role of N-Linked Glycosylation in Smoothened Signaling.

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Suresh Marada

2015-08-01

Full Text Available The G protein-coupled receptor (GPCR Smoothened (Smo is the requisite signal transducer of the evolutionarily conserved Hedgehog (Hh pathway. Although aspects of Smo signaling are conserved from Drosophila to vertebrates, significant differences have evolved. These include changes in its active sub-cellular localization, and the ability of vertebrate Smo to induce distinct G protein-dependent and independent signals in response to ligand. Whereas the canonical Smo signal to Gli transcriptional effectors occurs in a G protein-independent manner, its non-canonical signal employs Gαi. Whether vertebrate Smo can selectively bias its signal between these routes is not yet known. N-linked glycosylation is a post-translational modification that can influence GPCR trafficking, ligand responsiveness and signal output. Smo proteins in Drosophila and vertebrate systems harbor N-linked glycans, but their role in Smo signaling has not been established. Herein, we present a comprehensive analysis of Drosophila and murine Smo glycosylation that supports a functional divergence in the contribution of N-linked glycans to signaling. Of the seven predicted glycan acceptor sites in Drosophila Smo, one is essential. Loss of N-glycosylation at this site disrupted Smo trafficking and attenuated its signaling capability. In stark contrast, we found that all four predicted N-glycosylation sites on murine Smo were dispensable for proper trafficking, agonist binding and canonical signal induction. However, the under-glycosylated protein was compromised in its ability to induce a non-canonical signal through Gαi, providing for the first time evidence that Smo can bias its signal and that a post-translational modification can impact this process. As such, we postulate a profound shift in N-glycan function from affecting Smo ER exit in flies to influencing its signal output in mice.

Role of protein glycosylation on the expression of muscarinic receptors of N4TG1 neuroblastoma cells

International Nuclear Information System (INIS)

Ahmad, A.; Chiang, P.K.

1986-01-01

Muscarinic acetylcholine receptors (mAChR) are glycoproteins. Experiments were conducted to determine whether active glycosylation of proteins in N4TG1 neuroblastoma cells could affect the expression of muscarinic receptors on the cell surface. The binding of radioactive N-methylscopolamine, a membrane impermeable ligand, to intact cells was used as a measure of mAChR. In the presence of the inhibitors of glycosylation, such as tunicamycin, monensin and amphomycin, N-linked glycosylation of proteins in the N4TG1 cells was inhibited, as measured by the incorporation of radioactive glucosamine or mannose in proteins. At the concentrations of tunicamycin and monensin used, the glycosylation of proteins after 3 hours were drastically reduced, but the number of mAChR in the cells was not altered. The apparent lack of effect within a short incubation period could be attributed to the presence of preformed oligosaccharide dolichol readily available for N-glycosylation. However, after 24 hours, tunicamycin (0.05 μg/ml) caused a decrease in the number of mAChR by 17% without having any effect on protein synthesis. Therefore, de novo glycosylation of proteins may be required for the expression of mAChR receptors in the N4TG1 neuroblastoma cell surface

Functional relevance of protein glycosylation to the pro-inflammatory effects of extracellular matrix metalloproteinase inducer (EMMPRIN) on monocytes/macrophages.

Science.gov (United States)

Ge, Heng; Yuan, Wei; Liu, Jidong; He, Qing; Ding, Song; Pu, Jun; He, Ben

2015-01-01

Extracellular matrix metalloproteinase inducer (EMMPRIN) is an important pro-inflammatory protein involved in the cellular functions of monocytes/macrophages. We have hypothesized that high-level heterogeneousness of protein glycosylation of EMMPRIN may have functional relevance to its biological effects and affect the inflammatory activity of monocytes/macrophages. The glycosylation patterns of EMMPRIN expressed by monocytes/macrophages (THP-1 cells) in response to different extracellular stimuli were observed, and the structures of different glycosylation forms were identified. After the purification of highly- and less-glycosylated proteins respectively, the impacts of different glycosylation forms on the pro-inflammatory effects of EMMPRIN were examined in various aspects, such as cell adhesion to endothelial cells, cell migrations, cytokine expression, and activation of inflammatory signalling pathway. 1) It was mainly the highly-glycosylated form of EMMPRIN (HG-EMMPRIN) that increased after being exposed to inflammatory signals (PMA and H2O2). 2) Glycosylation of EMMPRIN in monocytes/macrophages led to N-linked-glycans being added to the protein, with the HG form containing complex-type glycans and the less-glycosylated form (LG) the simple type. 3) Only the HG-EMMPRIN but not the LG-EMMPRIN exhibited pro-inflammatory effects and stimulated inflammatory activities of the monocytes/macrophages (i.e., activation of ERK1/2 and NF-κB pathway, enhanced monocyte-endothelium adhesion, cell migration and matrix metalloproteinase -9 expression). Post-transcriptional glycosylation represents an important mechanism that determines the biological effects of EMMPRIN in monocytes/macrophages. Glycosylation of EMMPRIN may serve as a potential target for regulating the inflammatory activities of monocytes/macrophages.

Functional relevance of protein glycosylation to the pro-inflammatory effects of extracellular matrix metalloproteinase inducer (EMMPRIN on monocytes/macrophages.

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Heng Ge

Full Text Available Extracellular matrix metalloproteinase inducer (EMMPRIN is an important pro-inflammatory protein involved in the cellular functions of monocytes/macrophages. We have hypothesized that high-level heterogeneousness of protein glycosylation of EMMPRIN may have functional relevance to its biological effects and affect the inflammatory activity of monocytes/macrophages.The glycosylation patterns of EMMPRIN expressed by monocytes/macrophages (THP-1 cells in response to different extracellular stimuli were observed, and the structures of different glycosylation forms were identified. After the purification of highly- and less-glycosylated proteins respectively, the impacts of different glycosylation forms on the pro-inflammatory effects of EMMPRIN were examined in various aspects, such as cell adhesion to endothelial cells, cell migrations, cytokine expression, and activation of inflammatory signalling pathway.1 It was mainly the highly-glycosylated form of EMMPRIN (HG-EMMPRIN that increased after being exposed to inflammatory signals (PMA and H2O2. 2 Glycosylation of EMMPRIN in monocytes/macrophages led to N-linked-glycans being added to the protein, with the HG form containing complex-type glycans and the less-glycosylated form (LG the simple type. 3 Only the HG-EMMPRIN but not the LG-EMMPRIN exhibited pro-inflammatory effects and stimulated inflammatory activities of the monocytes/macrophages (i.e., activation of ERK1/2 and NF-κB pathway, enhanced monocyte-endothelium adhesion, cell migration and matrix metalloproteinase -9 expression.Post-transcriptional glycosylation represents an important mechanism that determines the biological effects of EMMPRIN in monocytes/macrophages. Glycosylation of EMMPRIN may serve as a potential target for regulating the inflammatory activities of monocytes/macrophages.

The C-terminal N-glycosylation sites of the human alpha1,3/4-fucosyltransferase III, -V, and -VI (hFucTIII, -V, adn -VI) are necessary for the expression of full enzyme activity.

Science.gov (United States)

Christensen, L L; Jensen, U B; Bross, P; Orntoft, T F

2000-09-01

The alpha1,3/4-fucosyltransferases are involved in the synthesis of fucosylated cell surface glycoconjugates. Human alpha1,3/4-fucosyltransferase III, -V, and -VI (hFucTIII, -V, and -VI) contain two conserved C-terminal N-glycosylation sites (hFucTIII: Asn154 and Asn185; hFucTV: Asn167 and Asn198; and hFucTVI: Asn153 and Asn184). In the present study, we have analyzed the functional role of these potential N-glycosylation sites, laying the main emphasis on the sites in hFucTIII. Tunicamycin treatment completely abolished hFucTIII enzyme activity while castanospermine treatment diminished hFucTIII enzyme activity to approximately 40% of the activity of the native enzyme. To further analyze the role of the conserved N-glycosylation sites in hFucTIII, -V, and -VI, we made a series of mutant genomic DNAs in which the asparagine residues in the potential C-terminal N-glycosylation sites were replaced by glutamine. Subsequently, the hFucTIII, -V, and -VI wild type and the mutants were expressed in COS-7 cells. All the mutants exhibited lower enzyme activity than the wild type and elimination of individual sites had different effects on the activity. The mutations did not affect the protein level of the mutants in the cells, but reduced the molecular mass as predicted. Kinetic analysis of hFucTIII revealed that lack of glycosylation at Asn185 did not change the Km values for the oligosaccharide acceptor and the nucleotide sugar donor. The present study demonstrates that hFucTIII, -V, and -VI require N-glycosylation at the two conserved C-terminal N-glycosylation sites for expression of full enzyme activity.

Functional importance of PAI-1 glycosylation

DEFF Research Database (Denmark)

Christensen, Anni; Naessens, Dominik; Skottrup, Peter

2001-01-01

Structure-function studies of plasminogen activator inhibitor-1 (PAI-1) have previously been performed mostly with non-glycosylated material expressed in E. coli. We have now studied the importance of PAI-1 glycosylation for its functional properties. PAI-1 has 3 potential sites for N......-glycosylated PAI-1 could be conferred upon PAI-1 expressed in HEK293 cells by mutational inactivation of one or the other glycosylation site. These findings reveal a novel functional role for glycosylation of a serpin. The glycosylation sites are localised between a-helix H and b-strand 2C and b-strand 3C and a...

N-Glycosylation of cholera toxin B subunit: serendipity for novel plant-made vaccines?

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Nobuyuki eMatoba

2015-12-01

Full Text Available The non-toxic B subunit of cholera toxin (CTB has attracted considerable interests from vaccinologists due to strong mucosal immunomodulatory effects and potential utility as a vaccine scaffold for heterologous antigens. Along with other conventional protein expression systems, various plant species have been used as recombinant production hosts for CTB and its fusion proteins. However, it has recently become clear that the protein is N-glycosylated within the endoplasmic reticulum of plant cells – a eukaryotic post-translational modification that is not present in native CTB. While functionally active aglycosylated variants have been successfully engineered to circumvent potential safety and regulatory issues related to glycosylation, this modification may actually provide advantageous characteristics to the protein as a vaccine platform. Based on data from our recent studies, I discuss the unique features of N-glycosylated CTB produced in plants for the development of novel vaccines.

GlcNAc-1-P-transferase–tunicamycin complex structure reveals basis for inhibition of N-glycosylation

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Yoo, Jiho; Mashalidis, Ellene H.; Kuk, Alvin C. Y.; Yamamoto, Kazuki; Kaeser, Benjamin; Ichikawa, Satoshi; Lee, Seok-Yong

2018-02-19

N-linked glycosylation is a predominant post-translational modification of protein in eukaryotes, and its dysregulation is the etiology of several human disorders. The enzyme UDP-N-acetylglucosamine:dolichyl-phosphate N-acetylglucosaminephosphotransferase (GlcNAc-1-P-transferase or GPT) catalyzes the first and committed step of N-linked glycosylation in the endoplasmic reticulum membrane, and it is the target of the natural product tunicamycin. Tunicamycin has potent antibacterial activity, inhibiting the bacterial cell wall synthesis enzyme MraY, but its usefulness as an antibiotic is limited by off-target inhibition of human GPT. Our understanding of how tunicamycin inhibits N-linked glycosylation and efforts to selectively target MraY are hampered by a lack of structural information. Here we present crystal structures of human GPT in complex with tunicamycin. In conclusion, structural and functional analyses reveal the difference between GPT and MraY in their mechanisms of inhibition by tunicamycin. We demonstrate that this difference could be exploited to design MraY-specific inhibitors as potential antibiotics.

Similarities and Differences in the Glycosylation Mechanisms in Prokaryotes and Eukaryotes

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Anne Dell

2010-01-01

Full Text Available Recent years have witnessed a rapid growth in the number and diversity of prokaryotic proteins shown to carry N- and/or O-glycans, with protein glycosylation now considered as fundamental to the biology of these organisms as it is in eukaryotic systems. This article overviews the major glycosylation pathways that are known to exist in eukarya, bacteria and archaea. These are (i oligosaccharyltransferase (OST-mediated N-glycosylation which is abundant in eukarya and archaea, but is restricted to a limited range of bacteria; (ii stepwise cytoplasmic N-glycosylation that has so far only been confirmed in the bacterial domain; (iii OST-mediated O-glycosylation which appears to be characteristic of bacteria; and (iv stepwise O-glycosylation which is common in eukarya and bacteria. A key aim of the review is to integrate information from the three domains of life in order to highlight commonalities in glycosylation processes. We show how the OST-mediated N- and O-glycosylation pathways share cytoplasmic assembly of lipid-linked oligosaccharides, flipping across the ER/periplasmic/cytoplasmic membranes, and transferring “en bloc” to the protein acceptor. Moreover these hallmarks are mirrored in lipopolysaccharide biosynthesis. Like in eukaryotes, stepwise O-glycosylation occurs on diverse bacterial proteins including flagellins, adhesins, autotransporters and lipoproteins, with O-glycosylation chain extension often coupled with secretory mechanisms.

The C-terminal N-glycosylation sites of the human α1,3/4-fucosyltransferase III, -V and -VI (hFucTIII, -V and -VI) are necessary for the expression of full enzyme activity

DEFF Research Database (Denmark)

Christensen, Lise Lotte; Jensen, Uffe Birk; Bross, Peter Gerd

2000-01-01

FucTIII enzyme activity to approximately 40% of the activity of the native enzyme. To further analyze the role of the conserved N-glycosylation sites in hFucTIII, -V, and -VI, we made a series of mutant genomic DNAs in which the asparagine residues in the potential C-terminal N-glycosylation sites were replaced...

In vivo production of non-glycosylated recombinant proteins in Nicotiana benthamiana plants by co-expression with Endo-β-N-acetylglucosaminidase H (Endo H) of Streptomyces plicatus

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Cicek, Kader; Gulec, Burcu; Ungor, Rifat; Hasanova, Gulnara

2017-01-01

A plant transient expression system, with eukaryotic post-translational modification machinery, offers superior efficiency, scalability, safety, and lower cost over other expression systems. However, due to aberrant N-glycosylation, this expression system may not be a suitable expression platform for proteins not carrying N-linked glycans in the native hosts. Therefore, it is crucial to develop a strategy to produce target proteins in a non-glycosylated form while preserving their native sequence, conformation and biological activity. Previously, we developed a strategy for enzymatic deglycosylation of proteins in planta by co-expressing bacterial peptide-N-glycosidase F (PNGase F). Though PNGase F removes oligosaccharides from glycosylated proteins, in so doing it causes an amino acid change due to the deamidation of asparagine to aspartate in the N-X-S/T site. Endo-β-N-acetylglucosaminidase (EC3.2.1.96, Endo H), another deglycosylating enzyme, catalyzes cleavage between two N-Acetyl-D-glucosamine residues of the chitobiose core of N-linked glycans, leaving a single N-Acetyl-D-glucosamine residue without the concomitant deamidation of asparagine. In this study, a method for in vivo deglycosylation of recombinant proteins in plants by transient co-expression with bacterial Endo H is described for the first time. Endo H was fully active in vivo. and successfully cleaved N-linked glycans from glycoproteins were tested. In addition, unlike the glycosylated form, in vivo Endo H deglycosylated Pfs48/45 was recognized by conformational specific Pfs48/45 monoclonal antibody, in a manner similar to its PNGase F deglycosylated counterpart. Furthermore, the deglycosylated PA83 molecule produced by Endo H showed better stability than a PNGase F deglycosylated counterpart. Thus, an Endo H in vivo deglycosylation approach provides another opportunity to develop vaccine antigens, therapeutic proteins, antibodies, and industrial enzymes. PMID:28827815

Characterization of the oligosaccharide structure of human glycosylated prolactin (G-hPRL) native and recombinant

International Nuclear Information System (INIS)

Marcos Vinicius Nucci Capone

2013-01-01

Human prolactin (hPRL) is a polypeptide hormone secreted by the anterior pituitary under the regulation of the hypothalamus, involved in a variety of biological processes such as mammary gland development and lactation. The recombinant product is important in medical diagnosis and treatment of failure of lactation. This hormone may occur in the form of non-glycosylated protein (NGhPRL) and glycosylated (G-hPRL) with molecular weights of approximately 23 and 25 kilodalton (kDa), respectively; has a single N-glycosylation site located at asparagine (Asn) position 31, which is partially occupied, thus being a particularly interesting model of glycosylation. The biological activity of G-hPRL is lower compared to NG-hPRL (~4 times) and its physiological function is not well defined: the portion of carbohydrate appears to have an important role in the hormone biosynthesis, secretion, biological activity, and plasma survival of the hormone. The main objective of this study was to compare the structures of N-glycans present in glycosylated pituitary prolactin (G-hPRL-NHPP) with those present in the recombinant. To obtain the recombinant G-hPRL the production was performed in laboratory scale from Chinese hamster ovary cells (CHO), genetically modified and adapted to growth in suspension. Cycloheximide (CHX), whose main effect was to increase the ratio G-hPRL/NG-hPRL from 5% to 38% was added to the culture medium, thereby facilitating the purification of G-hPRL. The G-hPRL was purified in two steps, a cation exchanger followed by a purification by reversed-phase high performance liquid chromatography (RP-HPLC) which demonstrated the efficient separation of the two isoforms of hPRL. Recombinant G-hPRL-IPEN was well characterized by several techniques confirming its purity and biological activity, including comparisons with other reference preparation of pituitary origin purchased from the N ational Hormone & Peptide Program (NHPPU. S.) . The composition of N-glycans present

Topological studies of hSVCT1, the human sodium-dependent vitamin C transporter and the influence of N-glycosylation on its intracellular targeting

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Velho, Albertina M. [Department of Biosciences University of Kent, CT2 7NJ (United Kingdom); Jarvis, Simon M., E-mail: S.M.Jarvis@westminster.ac.uk [Department of Biosciences University of Kent, CT2 7NJ (United Kingdom); University of Westminster, School of Biosciences, London W1W 6UW (United Kingdom)

2009-08-01

The Na{sup +}-dependent transporters, hSVCT1 and hSVCT2, were assessed in COS-1 cells for their membrane topology. Antibodies to N- and C-termini of hSVCT1 and C-terminus of hSVCT2 identified positive immunofluorescence only after permeabilisation, suggesting these regions are intracellular. PNGase F treatment confirmed that WT hSVCT1 ({approx} 70-100 kDa) is glycosylated and site-directed mutagenesis of the three putative N-glycosylation sites, Asn138, Asn144, Asn230, demonstrated that mutants N138Q and N144Q were glycosylated ({approx} 68-90 kDa) with only 31-65% of WT L-ascorbic acid (AA) uptake while the glycosylation profile of N230Q remained unaltered ({approx} 98% of WT activity). However, the N138Q/N144Q double mutant displayed barely detectable membrane expression at {approx} 65 kDa, no apparent glycosylation and minimal AA uptake (< 10%) with no discernible improvement in expression or activity when cultured at 28 {sup o}C or 37 {sup o}C. Marker protein immunocytochemistry with N138Q/N144Q identified intracellular aggregates with hSVCT1 localised at the nuclear membrane but absent at the plasma membrane thus implicating its role as a possible intracellular transporter and suggesting N-glycosylation is required for hSVCT1 membrane targeting. Also, Lys242 on the same putative hydrophilic loop as Asn230 after biotinylation was inaccessible from the extracellular side when analysed by MALDI-TOF MS. A new hSVCT1 secondary structure model supporting these findings is proposed.

N-Glycosylation of Lipocalin 2 Is Not Required for Secretion or Exosome Targeting

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Erawan Borkham-Kamphorst

2018-04-01

Full Text Available Lipocalin 2 (LCN2 is a highly conserved secreted adipokine acting as a serum transport protein for small hydrophobic molecules such as fatty acids and steroids. In addition, LCN2 limits bacterial growth by sequestering iron-containing siderophores and further protects against intestinal inflammation and tumorigenesis associated with alterations in the microbiota. Human LCN2 contains one N-glycosylation site conserved in other species. It was postulated that this post-translational modification could facilitate protein folding, protects from proteolysis, is required for proper trafficking from the Golgi apparatus to the cell surface, and might be relevant for effective secretion. We here show that the homologous nucleoside antibiotic tunicamycin blocks N-linked glycosylation but not secretion of LCN2 in primary murine hepatocytes, derivatives thereof, human lung carcinoma cell line A549, and human prostate cancer cell line PC-3. Moreover, both the glycosylated and the non-glycosylated LCN2 variants are equally targeted to exosomes, demonstrating that this post-translational modification is not necessary for proper trafficking of LCN2 into these membranous extracellular vesicles. Furthermore, a hydrophobic cluster analysis revealed that the N-glycosylation site is embedded in a highly hydrophobic evolutionarily conserved surrounding. In sum, our data indicate that the N-glycosylation of LCN2 is not required for proper secretion and exosome cargo recruitment in different cell types, but might be relevant to increase overall solubility.

Endoplasmic reticulum stress and N-glycosylation modulate expression of WFS1 protein

International Nuclear Information System (INIS)

Yamaguchi, Suguru; Ishihara, Hisamitsu; Tamura, Akira; Yamada, Takahiro; Takahashi, Rui; Takei, Daisuke; Katagiri, Hideki; Oka, Yoshitomo

2004-01-01

Mutations of the WFS1 gene are responsible for two hereditary diseases, Wolfram syndrome and low frequency sensorineural hearing loss. The WFS1 protein is a glycoprotein located in the endoplasmic reticulum (ER) membrane but its function is poorly understood. Herein we show WFS1 mRNA and protein levels in pancreatic islets to be increased with ER-stress inducers, thapsigargin and dithiothreitol. Another ER-stress inducer, the N-glycosylation inhibitor tunicamycin, also raised WFS1 mRNA but not protein levels. Site-directed mutagenesis showed both Asn-663 and Asn-748 to be N-glycosylated in mouse WFS1 protein. The glycosylation-defective WFS1 protein, in which Asn-663 and Asn-748 had been substituted with aspartate, exhibited an increased protein turnover rate. Consistent with this, the WFS1 protein was more rapidly degraded in the presence of tunicamycin. These data indicate that ER-stress and N-glycosylation play important roles in WFS1 expression and stability, and also suggest regulatory roles for this protein in ER-stress induced cell death

Mechanistic understanding of N-glycosylation in Ebola virus glycoprotein maturation and function.

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Wang, Bin; Wang, Yujie; Frabutt, Dylan A; Zhang, Xihe; Yao, Xiaoyu; Hu, Dan; Zhang, Zhuo; Liu, Chaonan; Zheng, Shimin; Xiang, Shi-Hua; Zheng, Yong-Hui

2017-04-07

The Ebola virus (EBOV) trimeric envelope glycoprotein (GP) precursors are cleaved into the receptor-binding GP 1 and the fusion-mediating GP 2 subunits and incorporated into virions to initiate infection. GP 1 and GP 2 form heterodimers that have 15 or two N -glycosylation sites (NGSs), respectively. Here we investigated the mechanism of how N -glycosylation contributes to GP expression, maturation, and function. As reported before, we found that, although GP 1 NGSs are not critical, the two GP 2 NGSs, Asn 563 and Asn 618 , are essential for GP function. Further analysis uncovered that Asn 563 and Asn 618 regulate GP processing, demannosylation, oligomerization, and conformation. Consequently, these two NGSs are required for GP incorporation into EBOV-like particles and HIV type 1 (HIV-1) pseudovirions and determine viral transduction efficiency. Using CRISPR/Cas9 technology, we knocked out the two classical endoplasmic reticulum chaperones calnexin (CNX) and/or calreticulin (CRT) and found that both CNX and CRT increase GP expression. Nevertheless, NGSs are not required for the GP interaction with CNX or CRT. Together, we conclude that, although Asn 563 and Asn 618 are not required for EBOV GP expression, they synergistically regulate its maturation, which determines its functionality. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

N-glycosylation of Colorectal Cancer Tissues

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Balog, Crina I. A.; Stavenhagen, Kathrin; Fung, Wesley L. J.; Koeleman, Carolien A.; McDonnell, Liam A.; Verhoeven, Aswin; Mesker, Wilma E.; Tollenaar, Rob A. E. M.; Deelder, André M.; Wuhrer, Manfred

2012-01-01

Colorectal cancer is the third most common cancer worldwide with an annual incidence of ∼1 million cases and an annual mortality rate of ∼655,000 individuals. There is an urgent need for identifying novel targets to develop more sensitive, reliable, and specific tests for early stage detection of colon cancer. Post-translational modifications are known to play an important role in cancer progression and immune surveillance of tumors. In the present study, we compared the N-glycan profiles from 13 colorectal cancer tumor tissues and corresponding control colon tissues. The N-glycans were enzymatically released, purified, and labeled with 2-aminobenzoic acid. Aliquots were profiled by hydrophilic interaction liquid chromatography (HILIC-HPLC) with fluorescence detection and by negative mode MALDI-TOF-MS. Using partial least squares discriminant analysis to investigate the N-glycosylation changes in colorectal cancer, an excellent separation and prediction ability were observed for both HILIC-HPLC and MALDI-TOF-MS data. For structure elucidation, information from positive mode ESI-ion trap-MS/MS and negative mode MALDI-TOF/TOF-MS was combined. Among the features with a high separation power, structures containing a bisecting GlcNAc were found to be decreased in the tumor, whereas sulfated glycans, paucimannosidic glycans, and glycans containing a sialylated Lewis type epitope were shown to be increased in tumor tissues. In addition, core-fucosylated high mannose N-glycans were detected in tumor samples. In conclusion, the combination of HILIC and MALDI-TOF-MS profiling of N-glycans with multivariate statistical analysis demonstrated its potential for identifying N-glycosylation changes in colorectal cancer tissues and provided new leads that might be used as candidate biomarkers. PMID:22573871

N-Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

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Niu, Canfang; Luo, Huiying; Shi, Pengjun; Huang, Huoqing; Wang, Yaru; Yang, Peilong

2015-01-01

N-Glycosylation can modulate enzyme structure and function. In this study, we identified two pepsin-resistant histidine acid phosphatase (HAP) phytases from Yersinia kristensenii (YkAPPA) and Yersinia rohdei (YrAPPA), each having an N-glycosylation motif, and one pepsin-sensitive HAP phytase from Yersinia enterocolitica (YeAPPA) that lacked an N-glycosylation site. Site-directed mutagenesis was employed to construct mutants by altering the N-glycosylation status of each enzyme, and the mutant and wild-type enzymes were expressed in Pichia pastoris for biochemical characterization. Compared with those of the N-glycosylation site deletion mutants and N-deglycosylated enzymes, all N-glycosylated counterparts exhibited enhanced pepsin resistance. Introduction of the N-glycosylation site into YeAPPA as YkAPPA and YrAPPA conferred pepsin resistance, shifted the pH optimum (0.5 and 1.5 pH units downward, respectively) and improved stability at acidic pH (83.2 and 98.8% residual activities at pH 2.0 for 1 h). Replacing the pepsin cleavage sites L197 and L396 in the immediate vicinity of the N-glycosylation motifs of YkAPPA and YrAPPA with V promoted their resistance to pepsin digestion when produced in Escherichia coli but had no effect on the pepsin resistance of N-glycosylated enzymes produced in P. pastoris. Thus, N-glycosylation may improve pepsin resistance by enhancing the stability at acidic pH and reducing pepsin's accessibility to peptic cleavage sites. This study provides a strategy, namely, the manipulation of N-glycosylation, for improvement of phytase properties for use in animal feed. PMID:26637601

N-glycosylation of the β2 adrenergic receptor regulates receptor function by modulating dimerization.

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Li, Xiaona; Zhou, Mang; Huang, Wei; Yang, Huaiyu

2017-07-01

N-glycosylation is a common post-translational modification of G-protein-coupled receptors (GPCRs). However, it remains unknown how N-glycosylation affects GPCR signaling. β 2 adrenergic receptor (β 2 AR) has three N-glycosylation sites: Asn6, Asn15 at the N-terminus, and Asn187 at the second extracellular loop (ECL2). Here, we show that deletion of the N-glycan did not affect receptor expression and ligand binding. Deletion of the N-glycan at the N-terminus rather than Asn187 showed decreased effects on isoproterenol-promoted G-protein-dependent signaling, β-arrestin2 recruitment, and receptor internalization. Both N6Q and N15Q showed decreased receptor dimerization, while N187Q did not influence receptor dimerization. As decreased β 2 AR homodimer accompanied with reduced efficiency for receptor function, we proposed that the N-glycosylation of β 2 AR regulated receptor function by influencing receptor dimerization. To verify this hypothesis, we further paid attention to the residues at the dimerization interface. Studies of Lys60 and Glu338, two residues at the receptor dimerization interface, exhibited that the K60A/E338A showed decreased β 2 AR dimerization and its effects on receptor signaling were similar to N6Q and N15Q, which further supported the importance of receptor dimerization for receptor function. This work provides new insights into the relationship among glycosylation, dimerization, and function of GPCRs. Peptide-N-glycosidase F (PNGase F, EC 3.2.2.11); endo-β-N-acetylglucosaminidase A (Endo-A, EC 3.2.1.96). © 2017 Federation of European Biochemical Societies.

Functional importance of PAI-1 glycosylation

DEFF Research Database (Denmark)

Christensen, Anni; Naessens, Dominik; Skottrup, Peter

susceptible PAI-1 variant was not necessarily the one used when raising the antibody. This and other observations indicated that the carbohydrate moieties or the glycosylation sites are unlikely to be part of the epitopes for these antibodies. The antibody susceptibility characteristic for non......Structure-function studies of plasminogen activator inhibitor-1 (PAI-1) have previously been performed mostly with non-glycosylated material expressed in E. coli. We have now studied the importance of PAI-1 glycosylation for its functional properties. PAI-1 has 3 potential sites for N......-linked glycosylation. Biochemical analysis of PAI-1 variants with substitutions of the Asn residues in each of these sites and expression in human embryonic kidney 293 (HEK293) cells showed that only Asn211 and Asn 267, but not Asn331 are glycosylated, and revealed a differential composition of the carbohydrate...

Kex1 protease is involved in yeast cell death induced by defective N-glycosylation, acetic acid, and chronological aging.

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Hauptmann, Peter; Lehle, Ludwig

2008-07-04

N-glycosylation in the endoplasmic reticulum is an essential protein modification and highly conserved in evolution from yeast to humans. The key step of this pathway is the transfer of the lipid-linked core oligosaccharide to the nascent polypeptide chain, catalyzed by the oligosaccharyltransferase complex. Temperature-sensitive oligosaccharyltransferase mutants of Saccharomyces cerevisiae at the restrictive temperature, such as wbp1-1, as well as wild-type cells in the presence of the N-glycosylation inhibitor tunicamycin display typical apoptotic phenotypes like nuclear condensation, DNA fragmentation, phosphatidylserine translocation, caspase-like activity, and reactive oxygen species accumulation. Since deletion of the yeast metacaspase YCA1 did not abrogate this death pathway, we postulated a different proteolytic process to be responsible. Here, we show that Kex1 protease is involved in the programmed cell death caused by defective N-glycosylation. Its disruption decreases caspase-like activity, production of reactive oxygen species, and fragmentation of mitochondria and, conversely, improves growth and survival of cells. Moreover, we demonstrate that Kex1 contributes also to the active cell death program induced by acetic acid stress or during chronological aging, suggesting that Kex1 plays a more general role in cellular suicide of yeast.

Encoding asymmetry of the N-glycosylation motif facilitates glycoprotein evolution.

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Ryan Williams

Full Text Available Protein N-glycosylation is found in all domains of life and has a conserved role in glycoprotein folding and stability. In animals, glycoproteins transit through the Golgi where the N-glycans are trimmed and rebuilt with sequences that bind lectins, an innovation that greatly increases structural diversity and redundancy of glycoprotein-lectin interaction at the cell surface. Here we ask whether the natural tension between increasing diversity (glycan-protein interactions and site multiplicity (backup and status quo might be revealed by a phylogenic examination of glycoproteins and NXS/T(X ≠ P N-glycosylation sites. Site loss is more likely by mutation at Asn encoded by two adenosine (A-rich codons, while site gain is more probable by generating Ser or Thr downstream of an existing Asn. Thus mutations produce sites at novel positions more frequently than the reversal of recently lost sites, and therefore more paths though sequence space are made available to natural selection. An intra-species comparison of secretory and cytosolic proteins revealed a departure from equilibrium in sequences one-mutation-away from NXS/T and in (A content, indicating strong selective pressures and exploration of N-glycosylation positions during vertebrate evolution. Furthermore, secretory proteins have evolved at rates proportional to N-glycosylation site number, indicating adaptive interactions between the N-glycans and underlying protein. Given the topology of the genetic code, mutation of (A is more often nonsynonomous, and Lys, another target of many PTMs, is also encoded by two (A-rich codons. An examination of acetyl-Lys sites in proteins indicated similar evolutionary dynamics, consistent with asymmetry of the target and recognition portions of modified sites. Our results suggest that encoding asymmetry is an ancient mechanism of evolvability that increases diversity and experimentation with PTM site positions. Strong selective pressures on PTMs may have

Mass spectrometry characterization for N-glycosylation of immunoglobulin Y from hen egg yolk.

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Sheng, Long; He, Zhenjiao; Liu, Yaping; Ma, Meihu; Cai, Zhaoxia

2018-03-01

Immunoglobulin Y (IgY) is a new therapeutic antibody that exists in hen egg yolk. It is a glycoprotein, not much is known about its N-glycan structures, site occupancy and site-specific N-glycosylation. In this study, purified protein from hen egg yolk was identified as IgY based on SDS-PAGE and MALDI-TOF/TOF MS. N-glycan was released from IgY using peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine-amidase treatment, and the molecular weight of IgY was calculated using the difference between the molecular weight of IgY and deglycosylated IgY. Two potential N-Glycosylation sites (ASN 308 and ASN 409 ) were detected on IgY by nanoLC-ESI MS. Sugar chains were separated using normal phase liquid chromatography after fluorescence labeling, and 17 N-glycan structures were confirmed using ESI-MS. The sugar chain pattern contained high-mannose oligosaccharide, hybrid oligosaccharide and complex oligosaccharide. These results could lead to other important information regarding IgY glycosylation. Copyright © 2017 Elsevier B.V. All rights reserved.

Human Plasma N-glycosylation as Analyzed by Matrix-Assisted Laser Desorption/Ionization-Fourier Transform Ion Cyclotron Resonance-MS Associates with Markers of Inflammation and Metabolic Health.

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Reiding, Karli R; Ruhaak, L Renee; Uh, Hae-Won; El Bouhaddani, Said; van den Akker, Erik B; Plomp, Rosina; McDonnell, Liam A; Houwing-Duistermaat, Jeanine J; Slagboom, P Eline; Beekman, Marian; Wuhrer, Manfred

2017-02-01

Glycosylation is an abundant co- and post-translational protein modification of importance to protein processing and activity. Although not template-defined, glycosylation does reflect the biological state of an organism and is a high-potential biomarker for disease and patient stratification. However, to interpret a complex but informative sample like the total plasma N-glycome, it is important to establish its baseline association with plasma protein levels and systemic processes. Thus far, large-scale studies (n >200) of the total plasma N-glycome have been performed with methods of chromatographic and electrophoretic separation, which, although being informative, are limited in resolving the structural complexity of plasma N-glycans. MS has the opportunity to contribute additional information on, among others, antennarity, sialylation, and the identity of high-mannose type species.Here, we have used matrix-assisted laser desorption/ionization (MALDI)-Fourier transform ion cyclotron resonance (FTICR)-MS to study the total plasma N-glycome of 2144 healthy middle-aged individuals from the Leiden Longevity Study, to allow association analysis with markers of metabolic health and inflammation. To achieve this, N-glycans were enzymatically released from their protein backbones, labeled at the reducing end with 2-aminobenzoic acid, and following purification analyzed by negative ion mode intermediate pressure MALDI-FTICR-MS. In doing so, we achieved the relative quantification of 61 glycan compositions, ranging from Hex 4 HexNAc 2 to Hex 7 HexNAc 6 dHex 1 Neu5Ac 4 , as well as that of 39 glycosylation traits derived thereof. Next to confirming known associations of glycosylation with age and sex by MALDI-FTICR-MS, we report novel associations with C-reactive protein (CRP), interleukin 6 (IL-6), body mass index (BMI), leptin, adiponectin, HDL cholesterol, triglycerides (TG), insulin, gamma-glutamyl transferase (GGT), alanine aminotransferase (ALT), and smoking. Overall

Glycosylation Helps Cellulase Enzymes Bind to Plant Cell Walls (Fact Sheet)

Energy Technology Data Exchange (ETDEWEB)

2012-06-01

Computer simulations suggest a new strategy to design enhanced enzymes for biofuels production. Large-scale computer simulations predict that the addition of glycosylation on carbohydrate-binding modules can dramatically improve the binding affinity of these protein domains over amino acid mutations alone. These simulations suggest that glycosylation can be used as a protein engineering tool to enhance the activity of cellulase enzymes, which are a key component in the conversion of cellulose to soluble sugars in the production of biofuels. Glycosylation is the covalent attachment of carbohydrate molecules to protein side chains, and is present in many proteins across all kingdoms of life. Moreover, glycosylation is known to serve a wide variety of functions in biological recognition, cell signaling, and metabolism. Cellulase enzymes, which are responsible for deconstructing cellulose found in plant cell walls to glucose, contain glycosylation that when modified can affect enzymatic activity-often in an unpredictable manner. To gain insight into the role of glycosylation on cellulase activity, scientists at the National Renewable Energy Laboratory (NREL) used computer simulation to predict that adding glycosylation on the carbohydrate-binding module of a cellulase enzyme dramatically boosts the binding affinity to cellulose-more than standard protein engineering approaches in which amino acids are mutated. Because it is known that higher binding affinity in cellulases leads to higher activity, this work suggests a new route to designing enhanced enzymes for biofuels production. More generally, this work suggests that tuning glycosylation in cellulase enzymes is a key factor to consider when engineering biochemical conversion processes, and that more work is needed to understand how glycosylation affects cellulase activity at the molecular level.

An enzymatic deglycosylation scheme enabling identification of core fucosylated N-glycans and O-glycosylation site mapping of human plasma proteins

DEFF Research Database (Denmark)

Hägglund, Per; Matthiesen, R.; Elortza, F.

2007-01-01

and N-acetyl-β-glucosaminidase) are also included. The two strategies were here applied to identify 103 N-glycosylation sites in the Cohn IV fraction of human plasma. In addition, Endo D/H digestion uniquely enabled identification of 23 fucosylated N-glycosylation sites. Several O-glycosylated peptides......, thereby reducing the complexity and facilitating glycosylation site determinations. Here, we have used two different enzymatic deglycosylation strategies for N-glycosylation site analysis. (1) Removal of entire N-glycan chains by peptide- N-glycosidase (PNGase) digestion, with concomitant deamidation...... of the released asparagine residue. The reaction is carried out in H218O to facilitate identification of the formerly glycosylated peptide by incorporatation of 18O into the formed aspartic acid residue. (2) Digestion with two endo-β- N-acetylglucosaminidases (Endo D and Endo H) that cleave the glycosidic bond...

Rapid chemical de-N-glycosylation and derivatization for liquid chromatography of immunoglobulin N-linked glycans.

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Akihiko Kameyama

Full Text Available Glycan analysis may result in exploitation of glycan biomarkers and evaluation of heterogeneity of glycosylation of biopharmaceuticals. For N-linked glycan analysis, we investigated alkaline hydrolysis of the asparagine glycosyl carboxamide of glycoproteins as a deglycosylation reaction. By adding hydroxylamine into alkaline de-N-glycosylation, we suppressed the degradation of released glycans and obtained a mixture of oximes, free glycans, and glycosylamines. The reaction was completed within 1 h, and the mixture containing oximes was easily tagged with 2-aminobenzamide by reductive amination. Here, we demonstrated N-linked glycan analysis using this method for a monoclonal antibody, and examined whether this method could liberate glycans without degradation from apo-transferrin containing NeuAc and NeuGc and horseradish peroxidase containing Fuc α1-3 GlcNAc at the reducing end. Furthermore, we compared glycan recoveries between conventional enzymatic glycan release and this method. Increasing the reaction temperature and reaction duration led to degradation, whereas decreasing these parameters resulted in lower release. Considering this balance, we proposed to carry out the reaction at 80°C for 1 h for asialo glycoproteins from mammals and at 50°C for 1 h for sialoglycoproteins.

Differential dependence on N-glycosylation of anthrax toxin receptors CMG2 and TEM8.

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Sarah Friebe

Full Text Available ANTXR 1 and 2, also known as TEM8 and CMG2, are two type I membrane proteins, which have been extensively studied for their role as anthrax toxin receptors, but with a still elusive physiological function. Here we have analyzed the importance of N-glycosylation on folding, trafficking and ligand binding of these closely related proteins. We find that TEM8 has a stringent dependence on N-glycosylation. The presence of at least one glycan on each of its two extracellular domains, the vWA and Ig-like domains, is indeed necessary for efficient trafficking to the cell surface. In the absence of any N-linked glycans, TEM8 fails to fold correctly and is recognized by the ER quality control machinery. Expression of N-glycosylation mutants reveals that CMG2 is less vulnerable to sugar loss. The absence of N-linked glycans in one of the extracellular domains indeed has little impact on folding, trafficking or receptor function of the wild type protein expressed in tissue culture cells. N-glycans do, however, seem required in primary fibroblasts from human patients. Here, the presence of N-linked sugars increases the tolerance to mutations in cmg2 causing the rare genetic disease Hyaline Fibromatosis Syndrome. It thus appears that CMG2 glycosylation provides a buffer towards genetic variation by promoting folding of the protein in the ER lumen.

Adaptive antibody diversification through N-linked glycosylation of the immunoglobulin variable region.

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van de Bovenkamp, Fleur S; Derksen, Ninotska I L; Ooijevaar-de Heer, Pleuni; van Schie, Karin A; Kruithof, Simone; Berkowska, Magdalena A; van der Schoot, C Ellen; IJspeert, Hanna; van der Burg, Mirjam; Gils, Ann; Hafkenscheid, Lise; Toes, René E M; Rombouts, Yoann; Plomp, Rosina; Wuhrer, Manfred; van Ham, S Marieke; Vidarsson, Gestur; Rispens, Theo

2018-02-20

A hallmark of B-cell immunity is the generation of a diverse repertoire of antibodies from a limited set of germline V(D)J genes. This repertoire is usually defined in terms of amino acid composition. However, variable domains may also acquire N -linked glycans, a process conditional on the introduction of consensus amino acid motifs ( N -glycosylation sites) during somatic hypermutation. High levels of variable domain glycans have been associated with autoantibodies in rheumatoid arthritis, as well as certain follicular lymphomas. However, the role of these glycans in the humoral immune response remains poorly understood. Interestingly, studies have reported both positive and negative effects on antibody affinity. Our aim was to elucidate the role of variable domain glycans during antigen-specific antibody responses. By analyzing B-cell repertoires by next-generation sequencing, we demonstrate that N -glycosylation sites are introduced at positions in which glycans can affect antigen binding as a result of a specific clustering of progenitor glycosylation sites in the germline sequences of variable domain genes. By analyzing multiple human monoclonal and polyclonal (auto)antibody responses, we subsequently show that this process is subject to selection during antigen-specific antibody responses, skewed toward IgG4, and positively contributes to antigen binding. Together, these results highlight a physiological role for variable domain glycosylation as an additional layer of antibody diversification that modulates antigen binding.

Proteomics and pathway analysis of N-glycosylated mammary gland proteins in response to Escherichia coli mastitis in cattle.

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Yang, Yongxin; Shen, Weijun; Zhao, Xiaowei; Zhao, Huiling; Huang, Dongwei; Cheng, Guanglong

2014-06-01

The aim of this study was to investigate the N-linked glycosylated protein profile of mammary tissue from healthy cows and cows with mastitis due to Escherichia coli, in order to understand the molecular mechanisms of the host response to mastitis. N-glycopeptides were enriched with a lectin mixture and identified through high-accuracy mass spectrometry. A total of 551 N-glycosylation sites, corresponding to 294 proteins, were identified in the mammary tissues of healthy cows; these glycoproteins were categorised into three functional groups and clustered into 11 specific pathways. A total of 511 N-glycosylation sites, corresponding to 283 glycosylated proteins, were detected in the mammary tissues of cows with E. coli mastitis. There were differences in N-glycosylation sites in 98 proteins in the mammary tissues of healthy cows and cows with mastitis due to E. coli. Most proteins with altered glycosylation were those involved in responses to stress, cell adhesion and the immune response, and were assigned to five specific pathways based on their gene ontology annotation. The results from this study show that the glycosylated protein profile in the mammary tissues of healthy and mastitic cows are different, and altered glycoproteins are associated with several pathways, including the lysosome and O-glycan biosynthesis pathways. Copyright © 2014. Published by Elsevier Ltd.

Ultrasound-Assisted Synthesis of 1-N-glycosyl-1H-1,2,3-Triazole Derivatives and their Anti-inflammatory Activity

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Gilson Bezerra Silva

2012-06-01

Full Text Available We have been synthesized various glycosyl triazoles from the reaction between glycosyl azide (1 and terminal alkynes (2a-g. The glycopiranosyl 1,2,3-triazoles (3-9 have been obtained in moderate-to-excellent yields (63-99% through the copper (I-catalyst 1,3-dipolar cycloaddition reaction at room temperature using ultrasound irradiation. In addition, preliminary anti-inflammatory tests have been performed in the compounds conjugates with benzoheterocycles (3-7 moieties that shown moderate activity

N-glycosylation increases the circulatory half-life of human growth hormone

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Flintegaard, Thomas V; Thygesen, Peter; Rahbek-Nielsen, Henrik

2010-01-01

Therapeutic use of recombinant GH typically involves daily sc injections. We examined the possibilities for prolonging the in vivo circulation of GH by introducing N-glycans. Human GH variants with a single potential N-glycosylation site (N-X-S/T) introduced by site-directed mutagenesis were expr...

Purification, cloning, characterization, and N-glycosylation analysis of a novel β-fructosidase from Aspergillus oryzae FS4 synthesizing levan- and neolevan-type fructooligosaccharides.

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Li Xu

Full Text Available β-Fructosidases are a widespread group of enzymes that catalyze the hydrolysis of terminal fructosyl units from various substrates. These enzymes also exhibit transglycosylation activity when they function with high concentrations of sucrose, which is used to synthesize fructooligosaccharides (FOS in the food industry. A β-fructosidase (BfrA with high transglycosylation activity was purified from Aspergillus oryzae FS4 as a monomeric glycoprotein. Compared with the most extensively studied Aspergillus spp. fructosidases that synthesize inulin-type β-(2-1-linked FOS, BfrA has unique transfructosylating property of synthesizing levan- and neolevan-type β-(2-6-linked FOS. The coding sequence (bfrAFS4, 1.86 kb of BfrA was amplified and expressed in Escherichia coli and Pichia pastoris. Both native and recombinant proteins showed transfructosylation and hydrolyzation activities with broad substrate specificity. These proteins could hydrolyze the following linkages: Glc α-1, 2-β Fru; Glc α-1, 3-α Fru; and Glc α-1, 5-β Fru. Compared with the unglycosylated E. coli-expressed BfrA (E.BfrA, the N-glycosylated native (N.BfrA and the P. pastoris-expressed BfrA (P.BfrA were highly stable at a wide pH range (pH 4 to 11, and significantly more thermostable at temperatures up to 50°C with a maximum activity at 55°C. Using sucrose as substrate, the Km and kcat values for total activity were 37.19±5.28 mM and 1.0016±0.039×104 s-1 for N.BfrA. Moreover, 10 of 13 putative N-glycosylation sites were glycosylated on N.BfrA, and N-glycosylation was essential for enzyme thermal stability and optima activity. Thus, BfrA has demonstrated as a well-characterized A. oryzae fructosidase with unique transfructosylating capability of synthesizing levan- and neolevan-type FOS.

Saccharomyces cerevisiae KTR4, KTR5 and KTR7 encode mannosyltransferases differentially involved in the N- and O-linked glycosylation pathways.

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Hernández, Nahúm V; López-Ramírez, Luz A; Díaz-Jiménez, Diana F; Mellado-Mojica, Erika; Martínez-Duncker, Iván; López, Mercedes G; Mora-Montes, Héctor M

2017-10-01

Saccharomyces cerevisiae is a model to understand basic aspects of protein glycosylation pathways. Although these metabolic routes have been thoroughly studied, there are still knowledge gaps; among them, the role of the MNT1/KRE2 gene family. This family is composed of nine members, with only six functionally characterized. The enzymes Ktr1, Ktr3, and Mnt1/Kre2 have overlapping activities in both O-linked and N-linked glycan synthesis; while Ktr2 and Yur1 participate exclusively in the elongation of the N-linked glycan outer chain. KTR6 encodes for a phosphomannosyltransferase that synthesizes the cell wall phosphomannan. Here, we aimed to establish the functional role of KTR4, KTR5 and KTR7 in the protein glycosylation pathways, by using heterologous complementation in Candida albicans null mutants lacking members of the MNT1/KRE2 gene family. The three S. cerevisiae genes restored defects in the C. albicans N-linked glycosylation pathway. KTR5 and KTR7 partially complemented a C. albicans null mutant with defects in the synthesis of O-linked glycans, and only KTR4 fully elongated the O-linked glycans like wild-type cells. Therefore, our results suggest that the three genes have a redundant activity in the S. cerevisiae N-linked glycosylation pathway, but KTR4 plays a major role in O-linked glycan synthesis. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Protein N-glycosylation in eukaryotic microalgae and its impact on the production of nuclear expressed biopharmaceuticals

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Elodie eMathieu-Rivet

2014-07-01

Full Text Available Microalgae are currently used for the production of food compounds. Recently, few microalgae species have been investigated as potential biofactories for the production of biopharmaceuticals. Indeed in this context, microalgae are cheap, classified as Generally Recognized As Safe (GRAS organisms and can be grown easily. However, problems remain to be solved before any industrial production of microalgae-made biopharmaceuticals. Among them, post-translational modifications of the proteins need to be considered. Especially, N-glycosylation acquired by the secreted recombinant proteins is of major concern since most of the biopharmaceuticals are N-glycosylated and it is well recognized that glycosylation represent one of their critical quality attribute. Therefore, the evaluation of microalgae as alternative cell factory for biopharmaceutical productions thus requires to investigate their N-glycosylation capability in order to determine to what extend it differs from their human counterpart and to determine appropriate strategies for remodelling the microalgae glycosylation into human-compatible oligosaccharides. Here, we review the secreted recombinant proteins which have been successfully produced in microalgae. We also report on recent bioinformatics and biochemical data concerning the structure of glycans N-linked to proteins from various microalgae phyla and comment the consequences on the glycan engineering strategies that may be necessary to render those microalgae-made biopharmaceuticals compatible with human therapy.

Identification of residues important for the activity of Haloferax volcanii AglD, a component of the archaeal N-glycosylation pathway.

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Kaminski, Lina; Eichler, Jerry

2010-05-06

In Haloferax volcanii, AglD adds the final hexose to the N-linked pentasaccharide decorating the S-layer glycoprotein. Not knowing the natural substrate of the glycosyltransferase, together with the challenge of designing assays compatible with hypersalinity, has frustrated efforts at biochemical characterization of AglD activity. To circumvent these obstacles, an in vivo assay designed to identify amino acid residues important for AglD activity is described. In the assay, restoration of AglD function in an Hfx. volcanii aglD deletion strain transformed to express plasmid-encoded versions of AglD, generated through site-directed mutagenesis at positions encoding residues conserved in archaeal homologues of AglD, is reflected in the behavior of a readily detectable reporter of N-glycosylation. As such Asp110 and Asp112 were designated as elements of the DXD motif of AglD, a motif that interacts with metal cations associated with nucleotide-activated sugar donors, while Asp201 was predicted to be the catalytic base of the enzyme.

Quantification of the N-glycosylated secretome by super-SILAC during breast cancer progression and in human blood Samples

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Boersema, P.J.; Geiger, T.; Wiśniewski, J.R.

2013-01-01

Cells secrete a large number of proteins to communicate with their surroundings. Furthermore, plasma membrane proteins and intracellular proteins can be released into the extracellular space by regulated or non-regulated processes. Here, we profiled the supernatant of 11 cell lines....... In total, 1398 unique N-glycosylation sites were identified and quantified. Enriching for N-glycosylated peptides focused the analysis on classically secreted and membrane proteins. N-glycosylated secretome profiles correctly clustered the different cell lines to their respective cancer stage, suggesting...

A Markov chain model for N-linked protein glycosylation – towards a low-parameter tool for model-driven glycoengineering

DEFF Research Database (Denmark)

Spahn, Philipp N.; Hansen, Anders Holmgaard; Hansen, Henning Gram

2016-01-01

Glycosylation is a critical quality attribute of most recombinant biotherapeutics. Consequently, drug development requires careful control of glycoforms to meet bioactivity and biosafety requirements. However, glycoengineering can be extraordinarily difficult given the complex reaction networks...... present a novel low-parameter approach to describe glycosylation using flux-balance and Markov chain modeling. The model recapitulates the biological complexity of glycosylation, but does not require user-provided kinetic information. We use this method to predict and experimentally validate glycoprofiles...

Human Plasma N-glycosylation as Analyzed by Matrix-Assisted Laser Desorption/Ionization-Fourier Transform Ion Cyclotron Resonance-MS Associates with Markers of Inflammation and Metabolic Health*

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Reiding, Karli R.; Ruhaak, L. Renee; Uh, Hae-Won; el Bouhaddani, Said; van den Akker, Erik B.; Plomp, Rosina; McDonnell, Liam A.; Houwing-Duistermaat, Jeanine J.; Slagboom, P. Eline; Beekman, Marian; Wuhrer, Manfred

2017-01-01

Glycosylation is an abundant co- and post-translational protein modification of importance to protein processing and activity. Although not template-defined, glycosylation does reflect the biological state of an organism and is a high-potential biomarker for disease and patient stratification. However, to interpret a complex but informative sample like the total plasma N-glycome, it is important to establish its baseline association with plasma protein levels and systemic processes. Thus far, large-scale studies (n >200) of the total plasma N-glycome have been performed with methods of chromatographic and electrophoretic separation, which, although being informative, are limited in resolving the structural complexity of plasma N-glycans. MS has the opportunity to contribute additional information on, among others, antennarity, sialylation, and the identity of high-mannose type species. Here, we have used matrix-assisted laser desorption/ionization (MALDI)-Fourier transform ion cyclotron resonance (FTICR)-MS to study the total plasma N-glycome of 2144 healthy middle-aged individuals from the Leiden Longevity Study, to allow association analysis with markers of metabolic health and inflammation. To achieve this, N-glycans were enzymatically released from their protein backbones, labeled at the reducing end with 2-aminobenzoic acid, and following purification analyzed by negative ion mode intermediate pressure MALDI-FTICR-MS. In doing so, we achieved the relative quantification of 61 glycan compositions, ranging from Hex4HexNAc2 to Hex7HexNAc6dHex1Neu5Ac4, as well as that of 39 glycosylation traits derived thereof. Next to confirming known associations of glycosylation with age and sex by MALDI-FTICR-MS, we report novel associations with C-reactive protein (CRP), interleukin 6 (IL-6), body mass index (BMI), leptin, adiponectin, HDL cholesterol, triglycerides (TG), insulin, gamma-glutamyl transferase (GGT), alanine aminotransferase (ALT), and smoking. Overall, the

Glycosylation analysis of recombinant neutral protease I from Aspergillus oryzae expressed in Pichia pastoris.

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Lei, Da; Xu, Yang; He, Qinghua; Pang, Yifeng; Chen, Bo; Xiong, Liang; Li, Yanping

2013-12-01

Neutral protease I from Aspergillus oryzae 3.042 was expressed in Pichia pastoris and its N-glycosylation properties were analyzed. After purification by nickel-affinity chromatography column, the recombinant neutral protease (rNPI) was confirmed to be N-glycosylated by periodicacid/Schiff's base staining and Endo H digestion. Moreover, the deglycosylated protein's molecular weight decreased to 43.3 kDa from 54.5 kDa analyzed by SDS-PAGE and MALDI-TOF-MS, and the hyperglycosylation extent was 21 %. The N-glycosylation site of rNPI was analyzed by nano LC-MS/MS after digesting by trypsin and Glu-C, and the unique potential site Asn41 of mature peptide was found to be glycosylated. Homology modeling of the 3D structure of rNPI indicated that the attached N-glycans hardly affected neutral protease's activity due to the great distance away from the active site of the enzyme.

Prediction of glycosylation sites using random forests

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Hirst Jonathan D

2008-11-01

Full Text Available Abstract Background Post translational modifications (PTMs occur in the vast majority of proteins and are essential for function. Prediction of the sequence location of PTMs enhances the functional characterisation of proteins. Glycosylation is one type of PTM, and is implicated in protein folding, transport and function. Results We use the random forest algorithm and pairwise patterns to predict glycosylation sites. We identify pairwise patterns surrounding glycosylation sites and use an odds ratio to weight their propensity of association with modified residues. Our prediction program, GPP (glycosylation prediction program, predicts glycosylation sites with an accuracy of 90.8% for Ser sites, 92.0% for Thr sites and 92.8% for Asn sites. This is significantly better than current glycosylation predictors. We use the trepan algorithm to extract a set of comprehensible rules from GPP, which provide biological insight into all three major glycosylation types. Conclusion We have created an accurate predictor of glycosylation sites and used this to extract comprehensible rules about the glycosylation process. GPP is available online at http://comp.chem.nottingham.ac.uk/glyco/.

Preventing E-cadherin aberrant N-glycosylation at Asn-554 improves its critical function in gastric cancer

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Carvalho, S; Catarino, TA; Dias, AM; Kato, M; Almeida, A; Hessling, B; Figueiredo, J; Gärtner, F; Sanches, JM; Ruppert, T; Miyoshi, E; Pierce, M; Carneiro, F; Kolarich, D; Seruca, R; Yamaguchi, Y; Taniguchi, N; Reis, CA; Pinho, SS

2016-01-01

E-cadherin is a central molecule in the process of gastric carcinogenesis and its posttranslational modifications by N-glycosylation have been described to induce a deleterious effect on cell adhesion associated with tumor cell invasion. However, the role that site-specific glycosylation of E-cadherin has in its defective function in gastric cancer cells needs to be determined. Using transgenic mice models and human clinical samples, we demonstrated that N-acetylglucosaminyltransferase V (GnT-V)-mediated glycosylation causes an abnormal pattern of E-cadherin expression in the gastric mucosa. In vitro models further indicated that, among the four potential N-glycosylation sites of E-cadherin, Asn-554 is the key site that is selectively modified with β1,6 GlcNAc-branched N-glycans catalyzed by GnT-V. This aberrant glycan modification on this specific asparagine site of E-cadherin was demonstrated to affect its critical functions in gastric cancer cells by affecting E-cadherin cellular localization, cis-dimer formation, molecular assembly and stability of the adherens junctions and cell–cell aggregation, which was further observed in human gastric carcinomas. Interestingly, manipulating this site-specific glycosylation, by preventing Asn-554 from receiving the deleterious branched structures, either by a mutation or by silencing GnT-V, resulted in a protective effect on E-cadherin, precluding its functional dysregulation and contributing to tumor suppression. PMID:26189796

Study of ethanol-induced Golgi disorganization reveals the potential mechanism of alcohol-impaired N-glycosylation

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Casey, Carol A.; Bhat, Ganapati; Holzapfel, Melissa S.; Petrosyan, Armen

2016-01-01

Background It is known that ethanol (EtOH) and its metabolites have a negative effect on protein glycosylation. The fragmentation of the Golgi apparatus induced by alteration of the structure of largest Golgi matrix protein, giantin, is the major consequence of damaging effects of EtOH-metabolism on the Golgi, however, the link between this and abnormal glycosylation remains unknown. Because previously we have shown that Golgi morphology dictates glycosylation, we examined the effect EtOH administration has on function of Golgi residential enzymes involved in N-glycosylation. Methods HepG2 cells transfected with mouse ADH1 (VA-13 cells) were treated with 35 mM ethanol for 72 h. Male Wistar rats were pair-fed Lieber-DeCarli diets for 5 to 8 weeks. Characterization of Golgi-associated mannosyl (α-1,3-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase (MGAT1), α-1,2-mannosidase (Man-I) and α-mannosidase II (Man-II) were performed in VA-13 cells and rat hepatocytes followed by 3D Structured Illumination Microscopy (SIM). Results First, we detected that EtOH administration results in the loss of sialylated N-glycans on asialoglycoprotein receptor, however the high mannose-type N-glycans are increased. Further analysis by 3D SIM microscopy revealed that EtOH treatment despite Golgi disorganization does not change cis-Golgi localization for Man-I, but does induce medial-to-cis relocation of MGAT1 and Man-II. Using different approaches, including electron microscopy, we revealed that EtOH treatment results in dysfunction of Arf1 GTPase followed by a deficiency in COPI vesicles at the Golgi. Silencing beta-COP or expression of GDP-bound mutant Arf1(T31N) mimics the EtOH effect on retaining MGAT1 and Man-II at the cis-Golgi, suggesting that (a) EtOH specifically blocks activation of Arf1, and (b) EtOH alters the proper localization of Golgi enzymes through impairment of COPI. Importantly, the level of MGAT1 was reduced, because likely MGAT1, contrary to Man-I and Man

Tissue transglutaminase inhibits the TRPV5-dependent calcium transport in an N-glycosylation-dependent manner

DEFF Research Database (Denmark)

Boros, Sandor; Xi, Qi; Dimke, Henrik Anthony

2011-01-01

Tissue transglutaminase (tTG) is a multifunctional Ca(2+)-dependent enzyme, catalyzing protein crosslinking. The transient receptor potential vanilloid (TRPV) family of cation channels was recently shown to contribute to the regulation of TG activities in keratinocytes and hence skin barrier form......, these observations imply that tTG is a novel extracellular enzyme inhibiting the activity of TRPV5. The inhibition of TRPV5 occurs in an N-glycosylation-dependent manner, signifying a common final pathway by which distinct extracellular factors regulate channel activity....

The S-Layer Glycoprotein of the Crenarchaeote Sulfolobus acidocaldarius Is Glycosylated at Multiple Sites with Chitobiose-Linked N-Glycans

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Elham Peyfoon

2010-01-01

Full Text Available Glycosylation of the S-layer of the crenarchaea Sulfolobus acidocaldarius has been investigated using glycoproteomic methodologies. The mature protein is predicted to contain 31 N-glycosylation consensus sites with approximately one third being found in the C-terminal domain spanning residues L1004-Q1395. Since this domain is rich in Lys and Arg and therefore relatively tractable to glycoproteomic analysis, this study has focused on mapping its N-glycosylation. Our analysis identified nine of the 11 consensus sequence sites, and all were found to be glycosylated. This constitutes a remarkably high glycosylation density in the C-terminal domain averaging one site for each stretch of 30–40 residues. Each of the glycosylation sites observed was shown to be modified with a heterogeneous family of glycans, with the largest having a composition Glc1Man2GlcNAc2 plus 6-sulfoquinovose (QuiS, consistent with the tribranched hexasaccharide previously reported in the cytochrome b558/566 of S. acidocaldarius. S. acidocaldarius is the only archaeal species whose N-glycans are known to be linked via the chitobiose core disaccharide that characterises the N-linked glycans of Eukarya.

Porcine dentin sialoprotein glycosylation and glycosaminoglycan attachments.

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Yamakoshi, Yasuo; Nagano, Takatoshi; Hu, Jan Cc; Yamakoshi, Fumiko; Simmer, James P

2011-02-03

Dentin sialophosphoprotein (Dspp) is a multidomain, secreted protein that is critical for the formation of tooth dentin. Mutations in DSPP cause inherited dentin defects categorized as dentin dysplasia type II and dentinogenesis imperfecta type II and type III. Dentin sialoprotein (Dsp), the N-terminal domain of dentin sialophosphoprotein (Dspp), is a highly glycosylated proteoglycan, but little is known about the number, character, and attachment sites of its carbohydrate moieties. To identify its carbohydrate attachment sites we isolated Dsp from developing porcine molars and digested it with endoproteinase Glu-C or pronase, fractionated the digestion products, identified fractions containing glycosylated peptides using a phenol sulfuric acid assay, and characterized the glycopeptides by N-terminal sequencing, amino acid analyses, or LC/MSMS. To determine the average number of sialic acid attachments per N-glycosylation, we digested Dsp with glycopeptidase A, labeled the released N-glycosylations with 2-aminobenzoic acid, and quantified the moles of released glycosylations by comparison to labeled standards of known concentration. Sialic acid was released by sialidase digestion and quantified by measuring β-NADH reduction of pyruvic acid, which was generated stoichiometrically from sialic acid by aldolase. To determine its forms, sialic acid released by sialidase digestion was labeled with 1,2-diamino-4,5-methyleneoxybenzene (DMB) and compared to a DMB-labeled sialic acid reference panel by RP-HPLC. To determine the composition of Dsp glycosaminoglycan (GAG) attachments, we digested Dsp with chondroitinase ABC and compared the chromotagraphic profiles of the released disaccharides to commercial standards. N-glycosylations were identified at Asn37, Asn77, Asn136, Asn155, Asn161, and Asn176. Dsp averages one sialic acid per N-glycosylation, which is always in the form of N-acetylneuraminic acid. O-glycosylations were tentatively assigned at Thr200, Thr216 and Thr

Porcine dentin sialoprotein glycosylation and glycosaminoglycan attachments

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Yamakoshi Fumiko

2011-02-01

Full Text Available Abstract Background Dentin sialophosphoprotein (Dspp is a multidomain, secreted protein that is critical for the formation of tooth dentin. Mutations in DSPP cause inherited dentin defects categorized as dentin dysplasia type II and dentinogenesis imperfecta type II and type III. Dentin sialoprotein (Dsp, the N-terminal domain of dentin sialophosphoprotein (Dspp, is a highly glycosylated proteoglycan, but little is known about the number, character, and attachment sites of its carbohydrate moieties. Results To identify its carbohydrate attachment sites we isolated Dsp from developing porcine molars and digested it with endoproteinase Glu-C or pronase, fractionated the digestion products, identified fractions containing glycosylated peptides using a phenol sulfuric acid assay, and characterized the glycopeptides by N-terminal sequencing, amino acid analyses, or LC/MSMS. To determine the average number of sialic acid attachments per N-glycosylation, we digested Dsp with glycopeptidase A, labeled the released N-glycosylations with 2-aminobenzoic acid, and quantified the moles of released glycosylations by comparison to labeled standards of known concentration. Sialic acid was released by sialidase digestion and quantified by measuring β-NADH reduction of pyruvic acid, which was generated stoichiometrically from sialic acid by aldolase. To determine its forms, sialic acid released by sialidase digestion was labeled with 1,2-diamino-4,5-methyleneoxybenzene (DMB and compared to a DMB-labeled sialic acid reference panel by RP-HPLC. To determine the composition of Dsp glycosaminoglycan (GAG attachments, we digested Dsp with chondroitinase ABC and compared the chromotagraphic profiles of the released disaccharides to commercial standards. N-glycosylations were identified at Asn37, Asn77, Asn136, Asn155, Asn161, and Asn176. Dsp averages one sialic acid per N-glycosylation, which is always in the form of N-acetylneuraminic acid. O-glycosylations were

The Emerging Importance of IgG Fab Glycosylation in Immunity.

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van de Bovenkamp, Fleur S; Hafkenscheid, Lise; Rispens, Theo; Rombouts, Yoann

2016-02-15

Human IgG is the most abundant glycoprotein in serum and is crucial for protective immunity. In addition to conserved IgG Fc glycans, ∼15-25% of serum IgG contains glycans within the variable domains. These so-called "Fab glycans" are primarily highly processed complex-type biantennary N-glycans linked to N-glycosylation sites that emerge during somatic hypermutation. Specific patterns of Fab glycosylation are concurrent with physiological and pathological conditions, such as pregnancy and rheumatoid arthritis. With respect to function, Fab glycosylation can significantly affect stability, half-life, and binding characteristics of Abs and BCRs. Moreover, Fab glycans are associated with the anti-inflammatory activity of IVIgs. Consequently, IgG Fab glycosylation appears to be an important, yet poorly understood, process that modulates immunity. Copyright © 2016 by The American Association of Immunologists, Inc.

The human receptor for urokinase plasminogen activator. NH2-terminal amino acid sequence and glycosylation variants

DEFF Research Database (Denmark)

Behrendt, N; Rønne, E; Ploug, M

1990-01-01

-PA. The purified protein shows a single 55-60 kDa band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. It is a heavily glycosylated protein, the deglycosylated polypeptide chain comprising only 35 kDa. The glycosylated protein contains N-acetyl-D-glucosamine and sialic acid......, but no N-acetyl-D-galactosamine. Glycosylation is responsible for substantial heterogeneity in the receptor on phorbol ester-stimulated U937 cells, and also for molecular weight variations among various cell lines. The amino acid composition and the NH2-terminal amino acid sequence are reported...

N-linked glycosylation at Asn152 on CD147 affects protein folding and stability: promoting tumour metastasis in hepatocellular carcinoma.

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Li, Jiang-Hua; Huang, Wan; Lin, Peng; Wu, Bo; Fu, Zhi-Guang; Shen, Hao-Miao; Jing, Lin; Liu, Zhen-Yu; Zhou, Yang; Meng, Yao; Xu, Bao-Qing; Chen, Zhi-Nan; Jiang, Jian-Li

2016-11-21

Cluster of differentiation 147 (CD147), also known as extracellular matrix metalloproteinase inducer, is a transmembrane glycoprotein that mediates oncogenic processes partly through N-glycosylation modifications. N-glycosylation has been demonstrated to be instrumental for the regulation of CD147 function during malignant transformation. However, the role that site-specific glycosylation of CD147 plays in its defective function in hepatocellular carcinomacells needs to be determined. Here, we demonstrate that the modification of N-glycosylation at Asn152 on CD147 strongly promotes hepatocellular carcinoma (HCC) invasion and migration. After the removal of N-glycans at Asn152, CD147 was more susceptible to degradation by ER-localized ubiquitin ligase-mediated endoplasmic reticulum-associated degradation (ERAD). Furthermore, N-linked glycans at Asn152 were required for CD147 to acquire and maintain proper folding in the ER. Moreover, N-linked glycans at Asn152 functioned as a recognition motif that was directly mediated by the CNX quality control system. Two phases in the retention-based ER chaperones system drove ER-localized CD147 trafficking to degradation. Deletion of N-linked glycosylation at Asn152 on CD147 significantly suppressed in situ tumour metastasis. These data could potentially shed light on the molecular regulation of CD147 through glycosylation and provide a valuable means of developing drugs that target N-glycans at Asn152 on CD147.

Modulation and modeling of monoclonal antibody N-linked glycosylation in mammalian cell perfusion reactors.

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Karst, Daniel J; Scibona, Ernesto; Serra, Elisa; Bielser, Jean-Marc; Souquet, Jonathan; Stettler, Matthieu; Broly, Hervé; Soos, Miroslav; Morbidelli, Massimo; Villiger, Thomas K

2017-09-01

Mammalian cell perfusion cultures are gaining renewed interest as an alternative to traditional fed-batch processes for the production of therapeutic proteins, such as monoclonal antibodies (mAb). The steady state operation at high viable cell density allows the continuous delivery of antibody product with increased space-time yield and reduced in-process variability of critical product quality attributes (CQA). In particular, the production of a confined mAb N-linked glycosylation pattern has the potential to increase therapeutic efficacy and bioactivity. In this study, we show that accurate control of flow rates, media composition and cell density of a Chinese hamster ovary (CHO) cell perfusion bioreactor allowed the production of a constant glycosylation profile for over 20 days. Steady state was reached after an initial transition phase of 6 days required for the stabilization of extra- and intracellular processes. The possibility to modulate the glycosylation profile was further investigated in a Design of Experiment (DoE), at different viable cell density and media supplement concentrations. This strategy was implemented in a sequential screening approach, where various steady states were achieved sequentially during one culture. It was found that, whereas high ammonia levels reached at high viable cell densities (VCD) values inhibited the processing to complex glycan structures, the supplementation of either galactose, or manganese as well as their synergy significantly increased the proportion of complex forms. The obtained experimental data set was used to compare the reliability of a statistical response surface model (RSM) to a mechanistic model of N-linked glycosylation. The latter outperformed the response surface predictions with respect to its capability and reliability in predicting the system behavior (i.e., glycosylation pattern) outside the experimental space covered by the DoE design used for the model parameter estimation. Therefore, we can

General N-and O-Linked Glycosylation of Lipoproteins in Mycoplasmas and Role of Exogenous Oligosaccharide.

Science.gov (United States)

Daubenspeck, James M; Jordan, David S; Simmons, Warren; Renfrow, Matthew B; Dybvig, Kevin

2015-01-01

The lack of a cell wall, flagella, fimbria, and other extracellular appendages and the possession of only a single membrane render the mycoplasmas structurally simplistic and ideal model organisms for the study of glycoconjugates. Most species have genomes of about 800 kb and code for few proteins predicted to have a role in glycobiology. The murine pathogens Mycoplasma arthritidis and Mycoplasma pulmonis have only a single gene annotated as coding for a glycosyltransferase but synthesize glycolipid, polysaccharide and glycoproteins. Previously, it was shown that M. arthritidis glycosylated surface lipoproteins through O-linkage. In the current study, O-linked glycoproteins were similarly found in M. pulmonis and both species of mycoplasma were found to also possess N-linked glycans at residues of asparagine and glutamine. Protein glycosylation occurred at numerous sites on surface-exposed lipoproteins with no apparent amino acid sequence specificity. The lipoproteins of Mycoplasma pneumoniae also are glycosylated. Glycosylation was dependent on the glycosidic linkages from host oligosaccharides. As far as we are aware, N-linked glycoproteins have not been previously described in Gram-positive bacteria, the organisms to which the mycoplasmas are phylogenetically related. The findings indicate that the mycoplasma cell surface is heavily glycosylated with implications for the modulation of mycoplasma-host interactions.

Identification of Residues Important for the Activity of Haloferax volcanii AglD, a Component of the Archaeal N-Glycosylation Pathway

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Lina Kaminski

2010-01-01

Full Text Available In Haloferax volcanii, AglD adds the final hexose to the N-linked pentasaccharide decorating the S-layer glycoprotein. Not knowing the natural substrate of the glycosyltransferase, together with the challenge of designing assays compatible with hypersalinity, has frustrated efforts at biochemical characterization of AglD activity. To circumvent these obstacles, an in vivo assay designed to identify amino acid residues important for AglD activity is described. In the assay, restoration of AglD function in an Hfx. volcanii aglD deletion strain transformed to express plasmid-encoded versions of AglD, generated through site-directed mutagenesis at positions encoding residues conserved in archaeal homologues of AglD, is reflected in the behavior of a readily detectable reporter of N-glycosylation. As such Asp110 and Asp112 were designated as elements of the DXD motif of AglD, a motif that interacts with metal cations associated with nucleotide-activated sugar donors, while Asp201 was predicted to be the catalytic base of the enzyme.

N-glycosylation-negative catalase: a useful tool for exploring the role of hydrogen peroxide in the endoplasmic reticulum.

Science.gov (United States)

Lortz, S; Lenzen, S; Mehmeti, I

2015-03-01

Disulfide bond formation during protein folding of nascent proteins is associated with the generation of H2O2 in the endoplasmic reticulum (ER). Approaches to quantifying H2O2 directly within the ER failed because of the oxidative environment in the ER lumen, and ER-specific catalase expression to detoxify high H2O2 concentrations resulted in an inactive protein owing to N-glycosylation. Therefore, the N-glycosylation motifs at asparagine-244 and -439 of the human catalase protein were deleted by site-directed mutagenesis. The ER-targeted expression of these variants revealed that the deletion of the N-glycosylation motif only at asparagine-244 (N244) was associated with the maintenance of full enzymatic activity in the ER. Expression of catalase N244 in the ER (ER-Catalase N244) was ER-specific and protected the cells significantly against exogenously added H2O2. With the expression of ER-Catalase N244, a highly effective H2O2 inactivation within the ER was achieved for the first time. Catalase has a high H2O2-inactivation capacity without the need of reducing cofactors, which might interfere with the ER redox homeostasis, and is not involved in protein folding. With these characteristics ER-Catalase N244 is an ideal tool to explore the impact of ER-generated H2O2 on the generation of disulfide bonds or to study the induction of ER-stress pathways through protein folding overload and accumulation of H2O2. Copyright © 2014 Elsevier Inc. All rights reserved.

Structure-function analysis of the human sialyltransferase ST3Gal I - Role of N-glycosylation and a novel conserved sialylmotif

DEFF Research Database (Denmark)

Jeanneau, C.; Chazalet, V.; Auge, C.

2004-01-01

of these residues and of the conserved residues of motif VS (HX4E) was assessed using as a template the human ST3Gal I. Mutational analysis showed that residues His(299) and Tyr(300) of the new motif, and His(316) of the VS motif, are essential for activity since their substitution by alanine yielded inactive...... showed that none of the mutants tested had any significant effect in nucleotide donor binding. Instead the mutant proteins were affected in their binding to the acceptor and/or demonstrated lower catalytic efficiency. Although the human ST3Gal I has four N-glycan attachment sites in its catalytic domain...... that are potentially glycosylated, none of them was shown to be necessary for enzyme activity. However, N-glycosylation appears to contribute to the proper folding and trafficking of the enzyme....

Analysis and metabolic engineering of lipid-linked oligosaccharides in glycosylation-deficient CHO cells

International Nuclear Information System (INIS)

Jones, Meredith B.; Tomiya, Noboru; Betenbaugh, Michael J.; Krag, Sharon S.

2010-01-01

Glycosylation-deficient Chinese Hamster Ovary (CHO) cell lines can be used to expand our understanding of N-glycosylation pathways and to study Congenital Disorders of Glycosylation, diseases caused by defects in the synthesis of N-glycans. The mammalian N-glycosylation pathway involves the step-wise assembly of sugars onto a dolichol phosphate (P-Dol) carrier, forming a lipid-linked oligosaccharide (LLO), followed by the transfer of the completed oligosaccharide onto the protein of interest. In order to better understand how deficiencies in this pathway affect the availability of the completed LLO donor for use in N-glycosylation, we used a non-radioactive, HPLC-based assay to examine the intermediates in the LLO synthesis pathway for CHO-K1 cells and for three different glycosylation-deficient CHO cell lines. B4-2-1 cells, which have a mutation in the dolichol phosphate-mannose synthase (DPM2) gene, accumulated LLO with the structure Man 5 GlcNAc 2 -P-P-Dol, while MI8-5 cells, which lack glucosyltransferase I (ALG6) activity, accumulated Man 9 GlcNAc 2 -P-P-Dol. CHO-K1 and MI5-4 cells both produced primarily the complete LLO, Glc 3 Man 9 GlcNAc 2 -P-P-Dol, though the relative quantity was lower in MI5-4. MI5-4 cells have reduced hexokinase activity which could affect the availability of many of the substrates required for LLO synthesis and, consequently, impair production of the final LLO donor. Increasing hexokinase activity by overexpressing hexokinase II in MI5-4 caused a decrease in the relative quantities of the incomplete LLO intermediates from Man 5 GlcNAc 2 -PP-Dol through Glc 1 Man 9 GlcNAc 2 -PP-Dol, and an increase in the relative quantity of the final LLO donor, Glc 3 Man 9 GlcNAc 2 -P-P-Dol. This study suggests that metabolic engineering may be a useful strategy for improving LLO availability for use in N-glycosylation.

Analysis and metabolic engineering of lipid-linked oligosaccharides in glycosylation-deficient CHO cells

Energy Technology Data Exchange (ETDEWEB)

Jones, Meredith B., E-mail: mbauman7@jhu.edu [Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 3400 North Charles Street, Maryland Hall 221, Baltimore, MD 21218 (United States); Tomiya, Noboru, E-mail: ntomiya1@jhu.edu [Department of Biology, Johns Hopkins University, 3400 North Charles Street, Mudd Hall 104A, Baltimore, MD 21218 (United States); Betenbaugh, Michael J., E-mail: beten@jhu.edu [Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 3400 North Charles Street, Maryland Hall 221, Baltimore, MD 21218 (United States); Krag, Sharon S., E-mail: skrag@jhsph.edu [Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, 615 North Wolfe Street, Baltimore, MD 21205 (United States)

2010-04-23

Glycosylation-deficient Chinese Hamster Ovary (CHO) cell lines can be used to expand our understanding of N-glycosylation pathways and to study Congenital Disorders of Glycosylation, diseases caused by defects in the synthesis of N-glycans. The mammalian N-glycosylation pathway involves the step-wise assembly of sugars onto a dolichol phosphate (P-Dol) carrier, forming a lipid-linked oligosaccharide (LLO), followed by the transfer of the completed oligosaccharide onto the protein of interest. In order to better understand how deficiencies in this pathway affect the availability of the completed LLO donor for use in N-glycosylation, we used a non-radioactive, HPLC-based assay to examine the intermediates in the LLO synthesis pathway for CHO-K1 cells and for three different glycosylation-deficient CHO cell lines. B4-2-1 cells, which have a mutation in the dolichol phosphate-mannose synthase (DPM2) gene, accumulated LLO with the structure Man{sub 5}GlcNAc{sub 2}-P-P-Dol, while MI8-5 cells, which lack glucosyltransferase I (ALG6) activity, accumulated Man{sub 9}GlcNAc{sub 2}-P-P-Dol. CHO-K1 and MI5-4 cells both produced primarily the complete LLO, Glc{sub 3}Man{sub 9}GlcNAc{sub 2}-P-P-Dol, though the relative quantity was lower in MI5-4. MI5-4 cells have reduced hexokinase activity which could affect the availability of many of the substrates required for LLO synthesis and, consequently, impair production of the final LLO donor. Increasing hexokinase activity by overexpressing hexokinase II in MI5-4 caused a decrease in the relative quantities of the incomplete LLO intermediates from Man{sub 5}GlcNAc{sub 2}-PP-Dol through Glc{sub 1}Man{sub 9}GlcNAc{sub 2}-PP-Dol, and an increase in the relative quantity of the final LLO donor, Glc{sub 3}Man{sub 9}GlcNAc{sub 2}-P-P-Dol. This study suggests that metabolic engineering may be a useful strategy for improving LLO availability for use in N-glycosylation.

Importance of glycosylation on function of a potassium channel in neuroblastoma cells.

Directory of Open Access Journals (Sweden)

M K Hall

Full Text Available The Kv3.1 glycoprotein, a voltage-gated potassium channel, is expressed throughout the central nervous system. The role of N-glycans attached to the Kv3.1 glycoprotein on conducting and non-conducting functions of the Kv3.1 channel are quite limiting. Glycosylated (wild type, partially glycosylated (N220Q and N229Q, and unglycosylated (N220Q/N229Q Kv3.1 proteins were expressed and characterized in a cultured neuronal-derived cell model, B35 neuroblastoma cells. Western blots, whole cell current recordings, and wound healing assays were employed to provide evidence that the conducting and non-conducting properties of the Kv3.1 channel were modified by N-glycans of the Kv3.1 glycoprotein. Electrophoretic migration of the various Kv3.1 proteins treated with PNGase F and neuraminidase verified that the glycosylation sites were occupied and that the N-glycans could be sialylated, respectively. The unglycosylated channel favored a different whole cell current pattern than the glycoform. Further the outward ionic currents of the unglycosylated channel had slower activation and deactivation rates than those of the glycosylated Kv3.1 channel. These kinetic parameters of the partially glycosylated Kv3.1 channels were also slowed. B35 cells expressing glycosylated Kv3.1 protein migrated faster than those expressing partially glycosylated and much faster than those expressing the unglycosylated Kv3.1 protein. These results have demonstrated that N-glycans of the Kv3.1 glycoprotein enhance outward ionic current kinetics, and neuronal migration. It is speculated that physiological changes which lead to a reduction in N-glycan attachment to proteins will alter the functions of the Kv3.1 channel.

Glycation and transglutaminase mediated glycosylation of fish gelatin peptides with glucosamine enhance bioactivity.

Science.gov (United States)

Hong, Pui Khoon; Gottardi, Davide; Ndagijimana, Maurice; Betti, Mirko

2014-01-01

A mixture of novel glycopeptides from glycosylation between cold water fish skin gelatin hydrolysates and glucosamine (GlcN) via transglutaminase (TGase), as well as glycation between fish gelatin hydrolysate and GlcN were identified by their pattern of molecular distribution using MALDI-TOF-MS. Glycated/glycosylated hydrolysates showed superior bioactivity to their original hydrolysates. Alcalase-derived fish skin gelatin hydrolysate glycosylated with GlcN in the presence of TGase at 25°C (FAT25) possessed antioxidant activity when tested in a linoleic acid oxidation system, when measured according to its 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity and when tested at the cellular level with human hepatocarcinoma (HepG2) cells as target cells. In addition, Alcalase-derived glycosylated hydrolysates showed specificity toward the inhibition of Escherichia coli (E. coli). The Flavourzyme-derived glycopeptides prepared at 37°C (FFC37 and FFT37) showed better DPPH scavenging activity than their native hydrolysates. The glycated Flavourzyme-derived hydrolysates were found to act as potential antimicrobial agents when incubated with E. coli and Bacillus subtilis. Copyright © 2013 Elsevier Ltd. All rights reserved.

Competition between folding and glycosylation in the endoplasmic reticulum

DEFF Research Database (Denmark)

Holst, B; Bruun, A W; Kielland-Brandt, Morten

1996-01-01

Using carboxypeptidase Y in Saccharomyces cerevisiae as a model system, the in vivo relationship between protein folding and N-glycosylation was studied. Seven new sites for N-glycosylation were introduced at positions buried in the folded protein structure. The level of glycosylation of such new...... acceptor sites. In some cases, all the newly synthesized mutant protein was modified at the novel site while in others no modification took place. In the most interesting category of mutants, the level of glycosylation was dependent on the conditions for folding. This shows that folding and glycosylation...

Glycosylation of voltage-gated calcium channels in health and disease

Czech Academy of Sciences Publication Activity Database

Lazniewska, Joanna; Weiss, Norbert

2017-01-01

Roč. 1859, č. 5 (2017), s. 662-668 ISSN 0005-2736 R&D Projects: GA ČR GA15-13556S; GA MŠk 7AMB15FR015 Institutional support: RVO:61388963 Keywords : calcium channels * voltage-gated calcium channels * N-glycosylation * ancillary subunit * trafficking * stability Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 3.498, year: 2016

Loci associated with N-glycosylation of human immunoglobulin G show pleiotropy with autoimmune diseases and haematological cancers

NARCIS (Netherlands)

Lauc, G.; Huffman, J.E.; Pucic, M.; Zgaga, L.; Adamczyk, B.; Muzinic, A.; Novokmet, M.; Polasek, O.; Gornik, O.; Kristic, J.; Keser, T.; Vitart, V.; Scheijen, B.; Uh, H.W.; Molokhia, M.; Patrick, A.L.; McKeigue, P.; Kolcic, I.; Lukic, I.K.; Swann, O.; Leeuwen, F.N. van; Ruhaak, L.R.; Houwing-Duistermaat, J.J.; Slagboom, P.E.; Beekman, M.; Craen, A.J. de; Deelder, A.M.; Zeng, Q.; Wang, W.; Hastie, N.D.; Gyllensten, U.; Wilson, J.F.; Wuhrer, M.; Wright, A.F.; Rudd, P.M.; Hayward, C.; Aulchenko, Y.; Campbell, H.; Rudan, I.

2013-01-01

Glycosylation of immunoglobulin G (IgG) influences IgG effector function by modulating binding to Fc receptors. To identify genetic loci associated with IgG glycosylation, we quantitated N-linked IgG glycans using two approaches. After isolating IgG from human plasma, we performed 77 quantitative

Yeast carboxypeptidase Y requires glycosylation for efficient intracellular transport, but not for vacuolar sorting, in vivo stability, or activity

DEFF Research Database (Denmark)

Winther, Jakob R.; Stevens, T H; Kielland-Brandt, Morten

1991-01-01

Functions of the carbohydrate side chains of the yeast vacuolar enzyme carboxypeptidase Y (CPY) were investigated by removal, through site-directed mutagenesis, of the sequences which act as signals for N-linked glycosylation. The mutant forms of the enzyme were analysed with respect to activity...

Evolutionary Pattern of N-Glycosylation Sequon Numbers  in Eukaryotic ABC Protein Superfamilies

Directory of Open Access Journals (Sweden)

R. Shyama Prasad Rao

2010-02-01

Full Text Available Many proteins contain a large number of NXS/T sequences (where X is any amino acid except proline which are the potential sites of asparagine (N linked glycosylation. However, the patterns of occurrence of these N-glycosylation sequons in related proteins or groups of proteins and their underlying causes have largely been unexplored. We computed the actual and probabilistic occurrence of NXS/T sequons in ABC protein superfamilies from eight diverse eukaryotic organisms. The ABC proteins contained significantly higher NXS/T sequon numbers compared to respective genome-wide average, but the sequon density was significantly lower owing to the increase in protein size and decrease in sequon specific amino acids. However, mammalian ABC proteins have significantly higher sequon density, and both serine and threonine containing sequons (NXS and NXT have been positively selected—against the recent findings of only threonine specific Darwinian selection of sequons in proteins. The occurrence of sequons was positively correlated with the frequency of sequon specific amino acids and negatively correlated with proline and the NPS/T sequences. Further, the NPS/T sequences were significantly higher than expected in plant ABC proteins which have the lowest number of NXS/T sequons. Accord- ingly, compared to overall proteins, N-glycosylation sequons in ABC protein superfamilies have a distinct pattern of occurrence, and the results are discussed in an evolutionary perspective.

Characterization of the oligosaccharide structure of human glycosylated prolactin (G-hPRL) native and recombinant; Caracterizacao da estrutura oligossacaridica de prolactina glicosilada humana (G-hPRL) nativa e recombinante

Energy Technology Data Exchange (ETDEWEB)

Marcos Vinicius Nucci Capone

2013-07-01

Human prolactin (hPRL) is a polypeptide hormone secreted by the anterior pituitary under the regulation of the hypothalamus, involved in a variety of biological processes such as mammary gland development and lactation. The recombinant product is important in medical diagnosis and treatment of failure of lactation. This hormone may occur in the form of non-glycosylated protein (NGhPRL) and glycosylated (G-hPRL) with molecular weights of approximately 23 and 25 kilodalton (kDa), respectively; has a single N-glycosylation site located at asparagine (Asn) position 31, which is partially occupied, thus being a particularly interesting model of glycosylation. The biological activity of G-hPRL is lower compared to NG-hPRL (~4 times) and its physiological function is not well defined: the portion of carbohydrate appears to have an important role in the hormone biosynthesis, secretion, biological activity, and plasma survival of the hormone. The main objective of this study was to compare the structures of N-glycans present in glycosylated pituitary prolactin (G-hPRL-NHPP) with those present in the recombinant. To obtain the recombinant G-hPRL the production was performed in laboratory scale from Chinese hamster ovary cells (CHO), genetically modified and adapted to growth in suspension. Cycloheximide (CHX), whose main effect was to increase the ratio G-hPRL/NG-hPRL from 5% to 38% was added to the culture medium, thereby facilitating the purification of G-hPRL. The G-hPRL was purified in two steps, a cation exchanger followed by a purification by reversed-phase high performance liquid chromatography (RP-HPLC) which demonstrated the efficient separation of the two isoforms of hPRL. Recombinant G-hPRL-IPEN was well characterized by several techniques confirming its purity and biological activity, including comparisons with other reference preparation of pituitary origin purchased from the {sup N}ational Hormone & Peptide Program (NHPPU. S.){sup .} The composition of N

Charge and Polarity Preferences for N-Glycosylation: A Genome-Wide In Silico Study and Its Implications Regarding Constitutive Proliferation and Adhesion of Carcinoma Cells.

Science.gov (United States)

Manwar Hussain, Muhammad Ramzan; Iqbal, Zeeshan; Qazi, Wajahat M; Hoessli, Daniel C

2018-01-01

The structural and functional diversity of the human proteome is mediated by N - and O- linked glycosylations that define the individual properties of extracellular and membrane-associated proteins. In this study, we utilized different computational tools to perform in silico based genome-wide mapping of 1,117 human proteins and unravel the contribution of both penultimate and vicinal amino acids for the asparagine-based, site-specific N -glycosylation. Our results correlate the non-canonical involvement of charge and polarity environment of classified amino acids (designated as L, O, A, P, and N groups) in the N -glycosylation process, as validated by NetNGlyc predictions, and 130 literature-reported human proteins. From our results, particular charge and polarity combinations of non-polar aliphatic, acidic, basic, and aromatic polar side chain environment of both penultimate and vicinal amino acids were found to promote the N -glycosylation process. However, the alteration in side-chain charge and polarity environment of genetic variants, particularly in the vicinity of Asn-containing epitope, may induce constitutive glycosylation (e.g., aberrant glycosylation at preferred and non-preferred sites) of membrane proteins causing constitutive proliferation and triggering epithelial-to-mesenchymal transition. The current genome-wide mapping of 1,117 proteins (2,909 asparagine residues) was used to explore charge- and polarity-based mechanistic constraints in N -glycosylation, and discuss alterations of the neoplastic phenotype that can be ascribed to N -glycosylation at preferred and non-preferred sites.

N-glycosylation of asparagine 8 regulates surface expression of major histocompatibility complex class I chain-related protein A (MICA) alleles dependent on threonine 24

DEFF Research Database (Denmark)

Pedersen, Maiken Mellergaard; Skovbakke, Sarah Line; Schneider, Christine L.

2014-01-01

for cell-surface expression and sought to identify the essential residues. We found that a single N-glycosylation site (N8) was important for MICA018 surface expression. The frequently expressed MICA allele 008, with an altered transmembrane and intracellular domain, was not affected by mutation of this N......-glycosylation site. Mutational analysis revealed that a single amino acid (T24) in the extracellular domain of MICA018 was essential for the N-glycosylation dependency, while the intracellular domain was not involved. The HHV7 immunoevasin, U21, was found to inhibit MICA018 surface expression by affecting N......-glycosylation and the retention was rescued by T24A substitution. Our study reveals N-glycosylation as an allele-specific regulatory mechanism important for regulation of surface expression of MICA018 and we pinpoint the residues essential for this N-glycosylation dependency. In addition we show that this regulatory mechanism...

Nutritional Therapies in Congenital Disorders of Glycosylation (CDG

Directory of Open Access Journals (Sweden)

Peter Witters

2017-11-01

Full Text Available Congenital disorders of glycosylation (CDG are a group of more than 130 inborn errors of metabolism affecting N-linked, O-linked protein and lipid-linked glycosylation. The phenotype in CDG patients includes frequent liver involvement, especially the disorders belonging to the N-linked protein glycosylation group. There are only a few treatable CDG. Mannose-Phosphate Isomerase (MPI-CDG was the first treatable CDG by high dose mannose supplements. Recently, with the successful use of d-galactose in Phosphoglucomutase 1 (PGM1-CDG, other CDG types have been trialed on galactose and with an increasing number of potential nutritional therapies. Current mini review focuses on therapies in glycosylation disorders affecting liver function and dietary intervention in general in N-linked glycosylation disorders. We also emphasize now the importance of early screening for CDG in patients with mild hepatopathy but also in cholestasis.

Biologically active, magnICON®-expressed EPO-Fc from stably transformed Nicotiana benthamiana plants presenting tetra-antennary N-glycan structures.

Science.gov (United States)

Nagels, Bieke; Van Damme, Els J M; Callewaert, Nico; Zabeau, Lennart; Tavernier, Jan; Delanghe, Joris R; Boets, Annemie; Castilho, Alexandra; Weterings, Koen

2012-08-31

In the past two decades plants have emerged as a valuable alternative for the production of pharmaceutical proteins. Since N-glycosylation influences functionality and stability of therapeutic proteins, the plant N-glycosylation pathway should be humanized. Here, we report the transient magnICON(®) expression of the erythropoietin fusion protein (EPO-Fc) in Nicotiana benthamiana plants that produce multi-antennary N-glycans without the plant-specific β1,2-xylose and α1,3-fucose residues in a stable manner (Nagels et al., 2011). The EPO-Fc fusion protein consists of EPO with a C-terminal-linked IgG-Fc domain and is used for pulmonary delivery of recombinant EPO to patients (Bitonti et al., 2004). Plant expressed EPO-Fc was quantified using a paramagnetic-particle chemiluminescent immunoassay and shown to be active in vitro via receptor binding experiments in HEK293T cells. Mass spectrometry-based N-glycan analysis confirmed the presence of multi-antennary N-glycans on plant-expressed EPO-Fc. The described research is the next step towards the development of a production platform for pharmaceutical proteins in plants. Copyright © 2012 Elsevier B.V. All rights reserved.

Glycosylation of KSHV Encoded vGPCR Functions in Its Signaling and Tumorigenicity

Directory of Open Access Journals (Sweden)

Hui Wu

2015-03-01

Full Text Available Kaposi’s sarcoma-associated herpesvirus (KSHV is a tumor virus and the etiologic agent of Kaposi’s Sarcoma (KS. KSHV G protein-coupled receptor (vGPCR is an oncogene that is implicated in malignancies associated with KHSV infection. In this study, we show that vGPCR undergoes extensive N-linked glycosylation within the extracellular domains, specifically asparagines 18, 22, 31 and 202. An immunofluorescence assay demonstrates that N-linked glycosylation are necessary for vGPCR trafficking to the cellular membrane. Employing vGPCR mutants whose glycosylation sites were ablated, we show that these vGPCR mutants failed to activate downstream signaling in cultured cells and were severely impaired to induce tumor formation in the xenograph nude mouse model. These findings support the conclusion that glycosylation is critical for vGPCR tumorigenesis and imply that chemokine regulation at the plasma membrane is crucial for vGPCR mediated signaling.

Links between CD147 Function, Glycosylation, and Caveolin-1

OpenAIRE

Tang, Wei; Chang, Sharon B.; Hemler, Martin E.

2004-01-01

Cell surface CD147 shows remarkable variations in size (31-65 kDa) because of heterogeneous N-glycosylation, with the most highly glycosylated forms functioning to induce matrix metalloproteinase (MMP) production. Here we show that all three CD147 N-glycosylation sites make similar contributions to both high and low glycoforms (HG- and LG-CD147). l-Phytohemagglutinin lectin binding and swainsonine inhibition experiments indicated that HG-CD147 contains N-acetylglucosaminyltransferase V-cataly...

Glycosylation of Recombinant Antigenic Proteins from Mycobacterium tuberculosis: In Silico Prediction of Protein Epitopes and Ex Vivo Biological Evaluation of New Semi-Synthetic Glycoconjugates.

Science.gov (United States)

Bavaro, Teodora; Tengattini, Sara; Piubelli, Luciano; Mangione, Francesca; Bernardini, Roberta; Monzillo, Vincenzina; Calarota, Sandra; Marone, Piero; Amicosante, Massimo; Pollegioni, Loredano; Temporini, Caterina; Terreni, Marco

2017-06-29

Tuberculosis is still one of the most deadly infectious diseases worldwide, and the use of conjugated antigens, obtained by combining antigenic oligosaccharides, such as the lipoarabinomannane (LAM), with antigenic proteins from Mycobacterium tuberculosis (MTB), has been proposed as a new strategy for developing efficient vaccines. In this work, we investigated the effect of the chemical glycosylation on two recombinant MTB proteins produced in E. coli with an additional seven-amino acid tag (recombinant Ag85B and TB10.4). Different semi-synthetic glycoconjugated derivatives were prepared, starting from mannose and two disaccharide analogs. The glycans were activated at the anomeric position with a thiocyanomethyl group, as required for protein glycosylation by selective reaction with lysines. The glycosylation sites and the ex vivo evaluation of the immunogenic activity of the different neo- glycoproteins were investigated. Glycosylation does not modify the immunological activity of the TB10.4 protein. Similarly, Ag85B maintains its B-cell activity after glycosylation while showing a significant reduction in the T-cell response. The results were correlated with the putative B- and T-cell epitopes, predicted using a combination of in silico systems. In the recombinant TB10.4, the unique lysine is not included in any T-cell epitope. Lys30 of Ag85B, identified as the main glycosylation site, proved to be the most important site involved in the formation of T-cell epitopes, reasonably explaining why its glycosylation strongly influenced the T-cell activity. Furthermore, additional lysines included in different epitopes (Lys103, -123 and -282) are also glycosylated. In contrast, B-cell epitopic lysines of Ag85B were found to be poorly glycosylated and, thus, the antibody interaction of Ag85B was only marginally affected after coupling with mono- or disaccharides.

Simple glycolipids of microbes: Chemistry, biological activity and metabolic engineering

Directory of Open Access Journals (Sweden)

Ahmad Mohammad Abdel-Mawgoud

2018-03-01

Full Text Available Glycosylated lipids (GLs are added-value lipid derivatives of great potential. Besides their interesting surface activities that qualify many of them to act as excellent ecological detergents, they have diverse biological activities with promising biomedical and cosmeceutical applications. Glycolipids, especially those of microbial origin, have interesting antimicrobial, anticancer, antiparasitic as well as immunomodulatory activities. Nonetheless, GLs are hardly accessing the market because of their high cost of production. We believe that experience of metabolic engineering (ME of microbial lipids for biofuel production can now be harnessed towards a successful synthesis of microbial GLs for biomedical and other applications. This review presents chemical groups of bacterial and fungal GLs, their biological activities, their general biosynthetic pathways and an insight on ME strategies for their production.

Comparative Proteomics and Glycoproteomics Reveal Increased N-Linked Glycosylation and Relaxed Sequon Specificity in Campylobacter jejuni NCTC11168 O

DEFF Research Database (Denmark)

Scott, Nichollas E.; Marzook, N. Bishara; Cain, Joel A.

2014-01-01

Campylobacter jejuni is a major cause of bacterial gastroenteritis. C. jejuni encodes a protein glycosylation (Pgl) locus responsible for the N-glycosylation of membrane-associated proteins. We examined two variants of the genome sequenced strain NCTC11168: O, a representative of the original...

A Systematic Study of Site-specific GalNAc-type O-Glycosylation Modulating Proprotein Convertase Processing

DEFF Research Database (Denmark)

Schjoldager, Katrine Ter-Borch Gram; Vester-Christensen, Malene B.; Goth, Christoffer K.

2011-01-01

Site-specific GalNAc-type O-glycosylation is emerging as an important co-regulator of proprotein convertase (PC) processing of proteins. PC processing is crucial in regulating many fundamental biological pathways and O-glycans in or immediately adjacent to processing sites may affect recognition...... and function of PCs. Thus, we previously demonstrated that deficiency in site-specific O-glycosylation in a PC site of the fibroblast growth factor, FGF23, resulted in marked reduction in secretion of active unprocessed FGF23, which cause familial tumoral calcinosis and hyperostosis hyperphosphatemia. GalNAc......-type O-glycosylation is found on serine and threonine amino acids and up to 20 distinct polypeptide GalNAc transferases catalyze the first addition of GalNAc to proteins making this step the most complex and differentially regulated steps in protein glycosylation. There is no reliable prediction model...

Label-free electrochemical biosensing of small-molecule inhibition on O-GlcNAc glycosylation.

Science.gov (United States)

Yang, Yu; Gu, Yuxin; Wan, Bin; Ren, Xiaomin; Guo, Liang-Hong

2017-09-15

O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) plays a critical role in modulating protein function in many cellular processes and human diseases such as Alzheimer's disease and type II diabetes, and has emerged as a promising new target. Specific inhibitors of OGT could be valuable tools to probe the biological functions of O-GlcNAcylation, but a lack of robust nonradiometric assay strategies to detect glycosylation, has impeded efforts to identify such compounds. Here we have developed a novel label-free electrochemical biosensor for the detection of peptide O-GlcNAcylation using protease-protection strategy and electrocatalytic oxidation of tyrosine mediated by osmium bipyridine as a signal reporter. There is a large difference in the abilities of proteolysis of the glycosylated and the unglycosylated peptides by protease, thus providing a sensing mechanism for OGT activity. When the O-GlcNAcylation is achieved, the glycosylated peptides cannot be cleaved by proteinase K and result in a high current response on indium tin oxide (ITO) electrode. However, when the O-GlcNAcylation is successfully inhibited using a small molecule, the unglycosylated peptides can be cleaved easily and lead to low current signal. Peptide O-GlcNAcylation reaction was performed in the presence of a well-defined small-molecule OGT inhibitor. The results indicated that the biosensor could be used to screen the OGT inhibitors effectively. Our label-free electrochemical method is a promising candidate for protein glycosylation pathway research in screening small-molecule inhibitors of OGT. Copyright © 2017 Elsevier B.V. All rights reserved.

Effect of glycosylation on biodistribution of radiolabeled glucagon-like peptide 1

International Nuclear Information System (INIS)

Watanabe, Ayahisa; Nishijima, Ken-ichi; Zhao, Songji; Tamaki, Nagara; Kuge, Yuji; Tanaka, Yoshikazu; Itoh, Takeshi; Takemoto, Hiroshi

2012-01-01

Glycosylation is generally applicable as a strategy for increasing the activity of bioactive proteins. In this study, we examined the effect of glycosylation on biodistribution of radiolabeled glucagon-like peptide 1 (GLP-1) as a bioactive peptide for type 2 diabetes. Noninvasive imaging studies were performed using a gamma camera after the intravenous administration of 123 I-GLP-1 or 123 I-α2, 6-sialyl N-acetyllactosamine (glycosylated) GLP-1 in rats. In ex vivo biodistribution studies using 125 I-GLP-1 or 125 I-glycosylated GLP-1, organ samples were measured for radioactivity. Plasma samples were added to 15% trichloroacetic acid (TCA) to obtain TCA-insoluble and TCA-soluble fractions. The radioactivity in the TCA-insoluble and TCA-soluble fractions was measured. In the noninvasive imaging studies, a relatively high accumulation level of 123 I-GLP-1 was found in the liver, which is the major organ to eliminate exogenous GLP-1. The area under the time-activity curve (AUC) of 123 I-glycosylated GLP-1 in the liver was significantly lower (89%) than that of 123 I-GLP-1. These results were consistent with those of ex vivo biodistribution studies using 125 I-labeled peptides. The AUC of 125 I-glycosylated GLP-1 in the TCA-insoluble fraction was significantly higher (1.7-fold) than that of GLP-1. This study demonstrated that glycosylation significantly decreased the distribution of radiolabeled GLP-1 into the liver and increased the concentration of radiolabeled GLP-1 in plasma. These results suggested that glycosylation is a useful strategy for decreasing the distribution into the liver of bioactive peptides as desirable pharmaceuticals. (author)

Glycosylation at Asn91 of H1N1 haemagglutinin affects binding to glycan receptors.

Science.gov (United States)

Jayaraman, Akila; Koh, Xiaoying; Li, Jing; Raman, Rahul; Viswanathan, Karthik; Shriver, Zachary; Sasisekharan, Ram

2012-06-15

The glycoprotein HA (haemagglutinin) on the surface of influenza A virus plays a central role in recognition and binding to specific host cell-surface glycan receptors and in fusion of viral membrane to the host nuclear membrane during viral replication. Given the abundance of HA on the viral surface, this protein is also the primary target for host innate and adaptive immune responses. Although addition of glycosylation sites on HA are a part of viral evolution to evade the host immune responses, there are specific glycosylation sites that are conserved during most of the evolution of the virus. In the present study, it was demonstrated that one such conserved glycosylation site at Asn(91) in H1N1 HA critically governs the glycan receptor-binding specificity and hence would potentially impinge on the host adaptation of the virus.

Trends and approaches in N-Glycosylation engineering in Chinese hamster ovary cell culture

DEFF Research Database (Denmark)

Fan, Yuzhou; Kildegaard, Helene Faustrup; Andersen, Mikael Rørdam

will summarize a group of recent strategies andapproaches and come up with case studies for N-glycosylation engineering in CHO cells and show several examples of relevantstudy cases from our research: 1) media and feed design, 2) culture process optimization, 3) substrate addition, 4) geneticengineering, 5...

Diagnostic accuracy of urinary prostate protein glycosylation profiling in prostatitis diagnosis.

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Vermassen, Tijl; Van Praet, Charles; Poelaert, Filip; Lumen, Nicolaas; Decaestecker, Karel; Hoebeke, Piet; Van Belle, Simon; Rottey, Sylvie; Delanghe, Joris

2015-01-01

Although prostatitis is a common male urinary tract infection, clinical diagnosis of prostatitis is difficult. The developmental mechanism of prostatitis is not yet unraveled which led to the elaboration of various biomarkers. As changes in asparagine-linked-(N-)-glycosylation were observed between healthy volunteers (HV), patients with benign prostate hyperplasia and prostate cancer patients, a difference could exist in biochemical parameters and urinary N-glycosylation between HV and prostatitis patients. We therefore investigated if prostatic protein glycosylation could improve the diagnosis of prostatitis. Differences in serum and urine biochemical markers and in total urine N-glycosylation profile of prostatic proteins were determined between HV (N=66) and prostatitis patients (N=36). Additionally, diagnostic accuracy of significant biochemical markers and changes in N-glycosylation was assessed. Urinary white blood cell (WBC) count enabled discrimination of HV from prostatitis patients (Pprostatitis patients from HV (Pprostatitis patients compared to HV (Pprostatitis. Further research is required to unravel the developmental course of prostatic inflammation.

Glycosylation site-targeted PEGylation of glucose oxidase retains native enzymatic activity.

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Ritter, Dustin W; Roberts, Jason R; McShane, Michael J

2013-04-10

Targeted PEGylation of glucose oxidase at its glycosylation sites was investigated to determine the effect on enzymatic activity, as well as the bioconjugate's potential in an optical biosensing assay. Methoxy-poly(ethylene glycol)-hydrazide (4.5kDa) was covalently coupled to periodate-oxidized glycosylation sites of glucose oxidase from Aspergillus niger. The bioconjugate was characterized using gel electrophoresis, liquid chromatography, mass spectrometry, and dynamic light scattering. Gel electrophoresis data showed that the PEGylation protocol resulted in a drastic increase (ca. 100kDa) in the apparent molecular mass of the protein subunit, with complete conversion to the bioconjugate; liquid chromatography data corroborated this large increase in molecular size. Mass spectrometry data proved that the extent of PEGylation was six poly(ethylene glycol) chains per glucose oxidase dimer. Dynamic light scattering data indicated the absence of higher-order oligomers in the PEGylated GOx sample. To assess stability, enzymatic activity assays were performed in triplicate at multiple time points over the course of 29 days in the absence of glucose, as well as before and after exposure to 5% w/v glucose for 24h. At a confidence level of 95%, the bioconjugate's performance was statistically equivalent to native glucose oxidase in terms of activity retention over the 29 day time period, as well as following the 24h glucose exposure. Finally, the bioconjugate was entrapped within a poly(2-hydroxyethyl methacrylate) hydrogel containing an oxygen-sensitive phosphor, and the construct was shown to respond approximately linearly with a 220±73% signal change (n=4, 95% confidence interval) over the physiologically-relevant glucose range (i.e., 0-400mg/dL); to our knowledge, this represents the first demonstration of PEGylated glucose oxidase incorporated into an optical biosensing assay. Copyright © 2013 Elsevier Inc. All rights reserved.

The impact of N-glycosylation on conformation and stability of immunoglobulin Y from egg yolk.

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Sheng, Long; He, Zhenjiao; Chen, Jiahui; Liu, Yaofa; Ma, Meihu; Cai, Zhaoxia

2017-03-01

Immunoglobulin Y (IgY) is a new therapeutic antibody, and its applications in industry are very broad. To provide insight into the effects of N-glycosylation on IgY, its conformation and stability were studied. In this research, IgY was extracted from egg yolk and then digested by peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine-amidase. SDS-PAGE and infrared absorption spectrum showed that carbohydrates were distinctly reduced after enzymolysis. The circular dichroism spectrum indicated that the IgY molecule became more flexible and disordered after removal of N-glycan. The fluorescence intensity revealed that Trp residues were buried in a more hydrophobic environment after disposal of N-glycan. Storage stability decreased with the removal of oligosaccharide chains based on size-exclusion chromatography analysis. Deglycosylated IgY exhibited less resistance to guanidine hydrochloride-induced unfolding. After deglycosylation, IgY was more sensitive to pepsin. Therefore, N-glycosylation played an important role in the maintenance of the structure and stability of IgY. Copyright © 2016 Elsevier B.V. All rights reserved.

The relative contribution of mannose salvage pathways to glycosylation in PMI-deficient mouse embryonic fibroblast cells.

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Fujita, Naonobu; Tamura, Ayako; Higashidani, Aya; Tonozuka, Takashi; Freeze, Hudson H; Nishikawa, Atsushi

2008-02-01

Mannose for mammalian glycan biosynthesis can be imported directly from the medium, derived from glucose or salvaged from endogenous or external glycans. All pathways must generate mannose 6-phosphate, the activated form of mannose. Imported or salvaged mannose is directly phosphorylated by hexokinase, whereas fructose 6-phosphate from glucose is converted to mannose 6-phosphate by phosphomannose isomerase (PMI). Normally, PMI provides the majority of mannose for glycan synthesis. To assess the contribution of PMI-independent pathways, we used PMI-null fibroblasts to study N-glycosylation of DNase I, a highly sensitive indicator protein. In PMI-null cells, imported mannose and salvaged mannose make a significant contribution to N-glycosylation. When these cells were grown in mannose-free medium along with the mannosidase inhibitor, swainsonine, to block the salvage pathways, N-glycosylation of DNase I was almost completely eliminated. Adding approximately 13 microm mannose to the medium completely restored normal glycosylation. Treatment with bafilomycin A(1), an inhibitor of lysosomal acidification, also markedly reduced N-glycosylation of DNase I, but in this case only 8 microm mannose was required to restore full glycosylation, indicating that a nonlysosomal source of mannose made a significant contribution. Glycosylation levels were greatly also reduced in glycoconjugate-free medium, when endosomal membrane trafficking was blocked by expression of a mutant SKD1. From these data, we conclude that PMI-null cells can salvage mannose from both endogenous and external glycoconjugates via lysosomal and nonlysosomal degradation pathways.

Trans-species Engineering of Glycosylated Therapeutic Proteins

DEFF Research Database (Denmark)

Yang, Zhang

important to address. Whenever glycosylation has been found to be an important PTM for function or bioactivity, human therapeutics have generally been produced in mammalian Chinese hamster ovary (CHO) cell line. Oglycosylation is one of the most complex regulated PTMs of proteins but also one of the least...... understood. Currently, mammalian cells are required for human O-glycosylation. Increasing efforts have been devoted to engineering non-mammalian cells for production of recombinant proteins with “human-like” glycosylation. Substantial success has been achieved with designed N-glycosylation in both lower......Recombinant expression of therapeutic proteins is one of the major tasks in modern biomedicine. One of the most important factors with respect to therapeutic use in human is posttranslational modifications (PTMs) of the recombinant proteins, of which protein glycosylation is by far the most...

Integrated proteomic and N-glycoproteomic analyses of doxorubicin sensitive and resistant ovarian cancer cells reveal glycoprotein alteration in protein abundance and glycosylation

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Hou, Junjie; Zhang, Chengqian; Xue, Peng; Wang, Jifeng; Chen, Xiulan; Guo, Xiaojing; Yang, Fuquan

2017-01-01

Ovarian cancer is one of the most common cancer among women in the world, and chemotherapy remains the principal treatment for patients. However, drug resistance is a major obstacle to the effective treatment of ovarian cancers and the underlying mechanism is not clear. An increased understanding of the mechanisms that underline the pathogenesis of drug resistance is therefore needed to develop novel therapeutics and diagnostic. Herein, we report the comparative analysis of the doxorubicin sensitive OVCAR8 cells and its doxorubicin-resistant variant NCI/ADR-RES cells using integrated global proteomics and N-glycoproteomics. A total of 1525 unique N-glycosite-containing peptides from 740 N-glycoproteins were identified and quantified, of which 253 N-glycosite-containing peptides showed significant change in the NCI/ADR-RES cells. Meanwhile, stable isotope labeling by amino acids in cell culture (SILAC) based comparative proteomic analysis of the two ovarian cancer cells led to the quantification of 5509 proteins. As about 50% of the identified N-glycoproteins are low-abundance membrane proteins, only 44% of quantified unique N-glycosite-containing peptides had corresponding protein expression ratios. The comparison and calibration of the N-glycoproteome versus the proteome classified 14 change patterns of N-glycosite-containing peptides, including 8 up-regulated N-glycosite-containing peptides with the increased glycosylation sites occupancy, 35 up-regulated N-glycosite-containing peptides with the unchanged glycosylation sites occupancy, 2 down-regulated N-glycosite-containing peptides with the decreased glycosylation sites occupancy, 46 down-regulated N-glycosite-containing peptides with the unchanged glycosylation sites occupancy. Integrated proteomic and N-glycoproteomic analyses provide new insights, which can help to unravel the relationship of N-glycosylation and multidrug resistance (MDR), understand the mechanism of MDR, and discover the new diagnostic and

Trans-species Engineering of Glycosylated Therapeutic Proteins

DEFF Research Database (Denmark)

Yang, Zhang

eukaryotes and even prokaryotes. Insect and yeast cells produce O-glycosylation incompatible with use in humans, however recently the yeast Pichia was engineered to perform the first step of human-like O-glycosylation. This review provides an overview of past and current engineering efforts of N...

Diversity in protein glycosylation among insect species.

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Gianni Vandenborre

Full Text Available BACKGROUND: A very common protein modification in multicellular organisms is protein glycosylation or the addition of carbohydrate structures to the peptide backbone. Although the Class of the Insecta is the largest animal taxon on Earth, almost all information concerning glycosylation in insects is derived from studies with only one species, namely the fruit fly Drosophila melanogaster. METHODOLOGY/PRINCIPAL FINDINGS: In this report, the differences in glycoproteomes between insects belonging to several economically important insect orders were studied. Using GNA (Galanthus nivalis agglutinin affinity chromatography, different sets of glycoproteins with mannosyl-containing glycan structures were purified from the flour beetle (Tribolium castaneum, the silkworm (Bombyx mori, the honeybee (Apis mellifera, the fruit fly (D. melanogaster and the pea aphid (Acyrthosiphon pisum. To identify and characterize the purified glycoproteins, LC-MS/MS analysis was performed. For all insect species, it was demonstrated that glycoproteins were related to a broad range of biological processes and molecular functions. Moreover, the majority of glycoproteins retained on the GNA column were unique to one particular insect species and only a few glycoproteins were present in the five different glycoprotein sets. Furthermore, these data support the hypothesis that insect glycoproteins can be decorated with mannosylated O-glycans. CONCLUSIONS/SIGNIFICANCE: The results presented here demonstrate that oligomannose N-glycosylation events are highly specific depending on the insect species. In addition, we also demonstrated that protein O-mannosylation in insect species may occur more frequently than currently believed.

Glycosylation patterns of kidney proteins differ in rat diabetic nephropathy.

Science.gov (United States)

Ravidà, Alessandra; Musante, Luca; Kreivi, Marjut; Miinalainen, Ilkka; Byrne, Barry; Saraswat, Mayank; Henry, Michael; Meleady, Paula; Clynes, Martin; Holthofer, Harry

2015-05-01

Diabetic nephropathy often progresses to end-stage kidney disease and, ultimately, to renal replacement therapy. Hyperglycemia per se is expected to have a direct impact on the biosynthesis of N- and O-linked glycoproteins. This study aims to establish the link between protein glycosylation and progression of experimental diabetic kidney disease using orthogonal methods. Kidneys of streptozotocin-diabetic and control rats were harvested at three different time points post streptozotocin injection. A panel of 12 plant lectins was used in the screening of lectin blots. The lectins UEAI, PHA-E, GSI, PNA, and RCA identified remarkable disease-associated differences in glycoprotein expression. Lectin affinity chromatography followed by mass spectrometric analyses led to the identification of several glycoproteins involved in salt-handling, angiogenesis, and extracellular matrix degradation. Our data confirm a substantial link between glycosylation signature and diabetes progression. Furthermore, as suggested by our findings on dipeptidyl peptidase-IV, altered protein glycosylation may reflect changes in biochemical properties such as enzymatic activity. Thus, our study demonstrates the unexplored potential of protein glycosylation analysis in the discovery of molecules linked to diabetic kidney disease.

Functional Analysis of Glycosylation of Zika Virus Envelope Protein

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Camila R. Fontes-Garfias

2017-10-01

Full Text Available Summary: Zika virus (ZIKV infection causes devastating congenital abnormities and Guillain-Barré syndrome. The ZIKV envelope (E protein is responsible for viral entry and represents a major determinant for viral pathogenesis. Like other flaviviruses, the ZIKV E protein is glycosylated at amino acid N154. To study the function of E glycosylation, we generated a recombinant N154Q ZIKV that lacks the E glycosylation and analyzed the mutant virus in mammalian and mosquito hosts. In mouse models, the mutant was attenuated, as evidenced by lower viremia, decreased weight loss, and no mortality; however, knockout of E glycosylation did not significantly affect neurovirulence. Mice immunized with the mutant virus developed a robust neutralizing antibody response and were completely protected from wild-type ZIKV challenge. In mosquitoes, the mutant virus exhibited diminished oral infectivity for the Aedes aegypti vector. Collectively, the results demonstrate that E glycosylation is critical for ZIKV infection of mammalian and mosquito hosts. : Zika virus (ZIKV causes devastating congenital abnormities and Guillain-Barré syndrome. Fontes-Garfias et al. showed that the glycosylation of ZIKV envelope protein plays an important role in infecting mosquito vectors and pathogenesis in mouse. Keywords: Zika virus, glycosylation, flavivirus entry, mosquito transmission, vaccine

Model-based analysis of N-glycosylation in Chinese hamster ovary cells

DEFF Research Database (Denmark)

Krambeck, Frederick J.; Bennun, Sandra V; Andersen, Mikael Rørdam

2017-01-01

The Chinese hamster ovary (CHO) cell is the gold standard for manufacturing of glycosylated recombinant proteins for production of biotherapeutics. The similarity of its glycosylation patterns to the human versions enable the products of this cell line favorable pharmacokinetic properties and lower...

Functional Analysis of Glycosylation of Zika Virus Envelope Protein.

Science.gov (United States)

Fontes-Garfias, Camila R; Shan, Chao; Luo, Huanle; Muruato, Antonio E; Medeiros, Daniele B A; Mays, Elizabeth; Xie, Xuping; Zou, Jing; Roundy, Christopher M; Wakamiya, Maki; Rossi, Shannan L; Wang, Tian; Weaver, Scott C; Shi, Pei-Yong

2017-10-31

Zika virus (ZIKV) infection causes devastating congenital abnormities and Guillain-Barré syndrome. The ZIKV envelope (E) protein is responsible for viral entry and represents a major determinant for viral pathogenesis. Like other flaviviruses, the ZIKV E protein is glycosylated at amino acid N154. To study the function of E glycosylation, we generated a recombinant N154Q ZIKV that lacks the E glycosylation and analyzed the mutant virus in mammalian and mosquito hosts. In mouse models, the mutant was attenuated, as evidenced by lower viremia, decreased weight loss, and no mortality; however, knockout of E glycosylation did not significantly affect neurovirulence. Mice immunized with the mutant virus developed a robust neutralizing antibody response and were completely protected from wild-type ZIKV challenge. In mosquitoes, the mutant virus exhibited diminished oral infectivity for the Aedes aegypti vector. Collectively, the results demonstrate that E glycosylation is critical for ZIKV infection of mammalian and mosquito hosts. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Biological significance of complex N-glycans in plants and their impact on plant physiology.

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Strasser, Richard

2014-01-01

Asparagine (N)-linked protein glycosylation is a ubiquitous co- and post-translational modification which can alter the biological function of proteins and consequently affects the development, growth, and physiology of organisms. Despite an increasing knowledge of N-glycan biosynthesis and processing, we still understand very little about the biological function of individual N-glycan structures in plants. In particular, the N-glycan-processing steps mediated by Golgi-resident enzymes create a structurally diverse set of protein-linked carbohydrate structures. Some of these complex N-glycan modifications like the presence of β1,2-xylose, core α1,3-fucose or the Lewis a-epitope are characteristic for plants and are evolutionary highly conserved. In mammals, complex N-glycans are involved in different cellular processes including molecular recognition and signaling events. In contrast, the complex N-glycan function is still largely unknown in plants. Here, in this short review, I focus on important recent developments and discuss their implications for future research in plant glycobiology and plant biotechnology.

Structure of the dimeric N-glycosylated form of fungal β-N-acetylhexosaminidase revealed by computer modeling, vibrational spectroscopy, and biochemical studies

Directory of Open Access Journals (Sweden)

Sklenář Jan

2007-05-01

Full Text Available Abstract Background Fungal β-N-acetylhexosaminidases catalyze the hydrolysis of chitobiose into its constituent monosaccharides. These enzymes are physiologically important during the life cycle of the fungus for the formation of septa, germ tubes and fruit-bodies. Crystal structures are known for two monomeric bacterial enzymes and the dimeric human lysosomal β-N-acetylhexosaminidase. The fungal β-N-acetylhexosaminidases are robust enzymes commonly used in chemoenzymatic syntheses of oligosaccharides. The enzyme from Aspergillus oryzae was purified and its sequence was determined. Results The complete primary structure of the fungal β-N-acetylhexosaminidase from Aspergillus oryzae CCF1066 was used to construct molecular models of the catalytic subunit of the enzyme, the enzyme dimer, and the N-glycosylated dimer. Experimental data were obtained from infrared and Raman spectroscopy, and biochemical studies of the native and deglycosylated enzyme, and are in good agreement with the models. Enzyme deglycosylated under native conditions displays identical kinetic parameters but is significantly less stable in acidic conditions, consistent with model predictions. The molecular model of the deglycosylated enzyme was solvated and a molecular dynamics simulation was run over 20 ns. The molecular model is able to bind the natural substrate – chitobiose with a stable value of binding energy during the molecular dynamics simulation. Conclusion Whereas the intracellular bacterial β-N-acetylhexosaminidases are monomeric, the extracellular secreted enzymes of fungi and humans occur as dimers. Dimerization of the fungal β-N-acetylhexosaminidase appears to be a reversible process that is strictly pH dependent. Oligosaccharide moieties may also participate in the dimerization process that might represent a unique feature of the exclusively extracellular enzymes. Deglycosylation had only limited effect on enzyme activity, but it significantly affected

Glycosylation differences contribute to distinct catalytic properties among bone alkaline phosphatase isoforms.

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Halling Linder, Cecilia; Narisawa, Sonoko; Millán, José Luis; Magnusson, Per

2009-11-01

Three circulating human bone alkaline phosphatase (BALP) isoforms (B1, B2, and B/I) can be distinguished in healthy individuals and a fourth isoform (B1x) has been discovered in patients with chronic kidney disease and in bone tissue. The present study was designed to correlate differing glycosylation patterns of each BALP isoform with their catalytic activity towards presumptive physiological substrates and to compare those properties with two recombinant isoforms of the tissue-nonspecific ALP (TNALP) isozyme, i.e., TNALP-flag, used extensively for mutation analysis of hypophosphatasia mutations and sALP-FcD(10), a chimeric enzyme recently used as therapeutic drug in a mouse model of infantile hypophosphatasia. The BALP isoforms were prepared from human osteosarcoma (SaOS-2) cells and the kinetic properties were evaluated using the synthetic substrate p-nitrophenylphosphate (pNPP) at pH 7.4 and 9.8, and the three suggested endogenous physiological substrates, i.e., inorganic pyrophosphate (PP(i)), pyridoxal 5'-phosphate (PLP), and phosphoethanolamine (PEA) at pH 7.4. Qualitative glycosylation differences were also assessed by lectin binding and precipitation. The k(cat)/K(M) was higher for B2 for all the investigated substrates. The catalytic activity towards PEA was essentially undetectable. The kinetic activity for TNALP-flag and sALP-FcD(10) was similar to the activity of the human BALP isoforms. The BALP isoforms differed in their lectin binding properties and dose-dependent lectin precipitation, which also demonstrated differences between native and denatured BALP isoforms. The observed differences in lectin specificity were attributed to N-linked carbohydrates. In conclusion, we demonstrate significantly different catalytic properties among the BALP isoforms due to structural differences in posttranslational glycosylation. Our data also suggests that PEA is not an endogenous substrate for the BALP isoforms or for the recombinant TNALP isoforms. The TNALP

Diversity and functions of protein glycosylation in insects.

Science.gov (United States)

Walski, Tomasz; De Schutter, Kristof; Van Damme, Els J M; Smagghe, Guy

2017-04-01

The majority of proteins is modified with carbohydrate structures. This modification, called glycosylation, was shown to be crucial for protein folding, stability and subcellular location, as well as protein-protein interactions, recognition and signaling. Protein glycosylation is involved in multiple physiological processes, including embryonic development, growth, circadian rhythms, cell attachment as well as maintenance of organ structure, immunity and fertility. Although the general principles of glycosylation are similar among eukaryotic organisms, insects synthesize a distinct repertoire of glycan structures compared to plants and vertebrates. Consequently, a number of unique insect glycans mediate functions specific to this class of invertebrates. For instance, the core α1,3-fucosylation of N-glycans is absent in vertebrates, while in insects this modification is crucial for the development of wings and the nervous system. At present, most of the data on insect glycobiology comes from research in Drosophila. Yet, progressively more information on the glycan structures and the importance of glycosylation in other insects like beetles, caterpillars, aphids and bees is becoming available. This review gives a summary of the current knowledge and recent progress related to glycan diversity and function(s) of protein glycosylation in insects. We focus on N- and O-glycosylation, their synthesis, physiological role(s), as well as the molecular and biochemical basis of these processes. Copyright © 2017 Elsevier Ltd. All rights reserved.

Immunoglobulin G (IgG) Fab glycosylation analysis using a new mass spectrometric high-throughput profiling method reveals pregnancy-associated changes.

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Bondt, Albert; Rombouts, Yoann; Selman, Maurice H J; Hensbergen, Paul J; Reiding, Karli R; Hazes, Johanna M W; Dolhain, Radboud J E M; Wuhrer, Manfred

2014-11-01

The N-linked glycosylation of the constant fragment (Fc) of immunoglobulin G has been shown to change during pathological and physiological events and to strongly influence antibody inflammatory properties. In contrast, little is known about Fab-linked N-glycosylation, carried by ∼ 20% of IgG. Here we present a high-throughput workflow to analyze Fab and Fc glycosylation of polyclonal IgG purified from 5 μl of serum. We were able to detect and quantify 37 different N-glycans by means of MALDI-TOF-MS analysis in reflectron positive mode using a novel linkage-specific derivatization of sialic acid. This method was applied to 174 samples of a pregnancy cohort to reveal Fab glycosylation features and their change with pregnancy. Data analysis revealed marked differences between Fab and Fc glycosylation, especially in the levels of galactosylation and sialylation, incidence of bisecting GlcNAc, and presence of high mannose structures, which were all higher in the Fab portion than the Fc, whereas Fc showed higher levels of fucosylation. Additionally, we observed several changes during pregnancy and after delivery. Fab N-glycan sialylation was increased and bisection was decreased relative to postpartum time points, and nearly complete galactosylation of Fab glycans was observed throughout. Fc glycosylation changes were similar to results described before, with increased galactosylation and sialylation and decreased bisection during pregnancy. We expect that the parallel analysis of IgG Fab and Fc, as set up in this paper, will be important for unraveling roles of these glycans in (auto)immunity, which may be mediated via recognition by human lectins or modulation of antigen binding. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Effects of cell culture conditions on antibody N-linked glycosylation--what affects high mannose 5 glycoform.

Science.gov (United States)

Pacis, Efren; Yu, Marcella; Autsen, Jennifer; Bayer, Robert; Li, Feng

2011-10-01

The glycosylation profile of therapeutic antibodies is routinely analyzed throughout development to monitor the impact of process parameters and to ensure consistency, efficacy, and safety for clinical and commercial batches of therapeutic products. In this study, unusually high levels of the mannose-5 (Man5) glycoform were observed during the early development of a therapeutic antibody produced from a Chinese hamster ovary (CHO) cell line, model cell line A. Follow up studies indicated that the antibody Man5 level was increased throughout the course of cell culture production as a result of increasing cell culture medium osmolality levels and extending culture duration. With model cell line A, Man5 glycosylation increased more than twofold from 12% to 28% in the fed-batch process through a combination of high basal and feed media osmolality and increased run duration. The osmolality and culture duration effects were also observed for four other CHO antibody producing cell lines by adding NaCl in both basal and feed media and extending the culture duration of the cell culture process. Moreover, reduction of Man5 level from model cell line A was achieved by supplementing MnCl2 at appropriate concentrations. To further understand the role of glycosyltransferases in Man5 level, N-acetylglucosaminyltransferase I GnT-I mRNA levels at different osmolality conditions were measured. It has been hypothesized that specific enzyme activity in the glycosylation pathway could have been altered in this fed-batch process. Copyright © 2011 Wiley Periodicals, Inc.

Prion propagation in cells expressing PrP glycosylation mutants.

Science.gov (United States)

Salamat, Muhammad K; Dron, Michel; Chapuis, Jérôme; Langevin, Christelle; Laude, Hubert

2011-04-01

Infection by prions involves conversion of a host-encoded cell surface protein (PrP(C)) to a disease-related isoform (PrP(Sc)). PrP(C) carries two glycosylation sites variably occupied by complex N-glycans, which have been suggested by previous studies to influence the susceptibility to these diseases and to determine characteristics of prion strains. We used the Rov cell system, which is susceptible to sheep prions, to generate a series of PrP(C) glycosylation mutants with mutations at one or both attachment sites. We examined their subcellular trafficking and ability to convert into PrP(Sc) and to sustain stable prion propagation in the absence of wild-type PrP. The susceptibility to infection of mutants monoglycosylated at either site differed dramatically depending on the amino acid substitution. Aglycosylated double mutants showed overaccumulation in the Golgi compartment and failed to be infected. Introduction of an ectopic glycosylation site near the N terminus fully restored cell surface expression of PrP but not convertibility into PrP(Sc), while PrP(C) with three glycosylation sites conferred cell permissiveness to infection similarly to the wild type. In contrast, predominantly aglycosylated molecules with nonmutated N-glycosylation sequons, produced in cells expressing glycosylphosphatidylinositol-anchorless PrP(C), were able to form infectious PrP(Sc). Together our findings suggest that glycosylation is important for efficient trafficking of anchored PrP to the cell surface and sustained prion propagation. However, properly trafficked glycosylation mutants were not necessarily prone to conversion, thus making it difficult in such studies to discern whether the amino acid changes or glycan chain removal most influences the permissiveness to prion infection.

Hydrophobic Man-1-P derivatives correct abnormal glycosylation in Type I congenital disorder of glycosylation fibroblasts.

Science.gov (United States)

Eklund, Erik A; Merbouh, Nabyl; Ichikawa, Mie; Nishikawa, Atsushi; Clima, Jessica M; Dorman, James A; Norberg, Thomas; Freeze, Hudson H

2005-11-01

Patients with Type I congenital disorders of glycosylation (CDG-I) make incomplete lipid-linked oligosaccharides (LLO). These glycans are poorly transferred to proteins resulting in unoccupied glycosylation sequons. Mutations in phosphomannomutase (PMM2) cause CDG-Ia by reducing the activity of PMM, which converts mannose (Man)-6-P to Man-1-P before formation of GDP-Man. These patients have reduced Man-1-P and GDP-Man. To replenish intracellular Man-1-P pools in CDG-Ia cells, we synthesized two hydrophobic, membrane permeable acylated versions of Man-1-P and determined their ability to normalize LLO size and N-glycosylation in CDG-Ia fibroblasts. Both compounds, compound I (diacetoxymethyl 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl phosphate) (C-I) and compound II (diacetoxymethyl 2,3,4,6-tetra-O-ethyloxycarbonyl-alpha-D-mannopyranosyl phosphate) (C-II), contain two acetoxymethyl (CH2OAc) groups O-linked to phosphorous. C-I contains acetyl esters and C-II contains ethylcarbonate (CO2Et) esters on the Man residue. Both C-I and C-II normalized truncated LLO, but C-II was about 2-fold more efficient than C-I. C-II replenished the GDP-Man pool in CDG-Ia cells and was more efficiently incorporated into glycoproteins than exogenous Man at low concentrations (25-75 mM). In a glycosylation assay of DNaseI in CDG-Ia cells, C-II restored glycosylation to control cell levels. C-II also corrected impaired LLO biosynthesis in cells from a Dolichol (Dol)-P-Man deficient patient (CDG-Ie) and partially corrected LLO in cells from an ALG12 mannosyltransferase-deficient patient (CDG-Ig), whereas cells from an ALG3-deficient patient (CDG-Id) and from an MPDU1-deficient patient (CDG-If) were not corrected. These results validate the general concept of using pro-Man-1-P substrates as potential therapeutics for CDG-I patients.

Optimal Synthetic Glycosylation of a Therapeutic Antibody.

Science.gov (United States)

Parsons, Thomas B; Struwe, Weston B; Gault, Joseph; Yamamoto, Keisuke; Taylor, Thomas A; Raj, Ritu; Wals, Kim; Mohammed, Shabaz; Robinson, Carol V; Benesch, Justin L P; Davis, Benjamin G

2016-02-12

Glycosylation patterns in antibodies critically determine biological and physical properties but their precise control is a significant challenge in biology and biotechnology. We describe herein the optimization of an endoglycosidase-catalyzed glycosylation of the best-selling biotherapeutic Herceptin, an anti-HER2 antibody. Precise MS analysis of the intact four-chain Ab heteromultimer reveals nonspecific, non-enzymatic reactions (glycation), which are not detected under standard denaturing conditions. This competing reaction, which has hitherto been underestimated as a source of side products, can now be minimized. Optimization allowed access to the purest natural form of Herceptin to date (≥90 %). Moreover, through the use of a small library of sugars containing non-natural functional groups, Ab variants containing defined numbers of selectively addressable chemical tags (reaction handles at Sia C1) in specific positions (for attachment of cargo molecules or "glycorandomization") were readily generated.

Influence of culture medium supplementation of tobacco NT1 cell suspension cultures on the N-glycosylation of human secreted alkaline phosphatase.

Science.gov (United States)

Becerra-Arteaga, Alejandro; Shuler, Michael L

2007-08-15

We report for the first time that culture conditions, specifically culture medium supplementation with nucleotide-sugar precursors, can alter significantly the N-linked glycosylation of a recombinant protein in plant cell culture. Human secreted alkaline phosphatase produced in tobacco NT1 cell suspension cultures was used as a model system. Plant cell cultures were supplemented with ammonia (30 mM), galactose (1 mM) and glucosamine (10 mM) to improve the extent of N-linked glycosylation. The highest levels of cell density and active extracellular SEAP in supplemented cultures were on average 260 g/L and 0.21 U/mL, respectively, compared to 340 g/L and 0.4 U/mL in unsupplemented cultures. The glycosylation profile of SEAP produced in supplemented cultures was determined via electrospray ionization mass spectrometry with precursor ion scanning and compared to that of SEAP produced in unsupplemented cultures. In supplemented and unsupplemented cultures, two biantennary complex-type structures terminated with one or two N-acetylglucosamines and one paucimannosidic glycan structure comprised about 85% of the SEAP glycan pool. These three structures contained plant-specific xylose and fucose residues and their relative abundances were affected by each supplement. High mannose structures (6-9 mannose residues) accounted for the remaining 15% glycans in all cases. The highest proportion (approximately 66%) of a single complex-type biantennary glycan structure terminated in both antennae by N- acetylglucosamine was obtained with glucosamine supplementation versus only 6% in unsupplemented medium. This structure is amenable for in vitro modification to yield a more human-like glycan and could serve as a route to plant cell culture produced therapeutic glycoproteins. (c) 2007 Wiley Periodicals, Inc.

Flagellar glycosylation in Clostridium botulinum.

Science.gov (United States)

Twine, Susan M; Paul, Catherine J; Vinogradov, Evgeny; McNally, David J; Brisson, Jean-Robert; Mullen, James A; McMullin, David R; Jarrell, Harold C; Austin, John W; Kelly, John F; Logan, Susan M

2008-09-01

Flagellins from Clostridium botulinum were shown to be post-translationally modified with novel glycan moieties by top-down MS analysis of purified flagellin protein from strains of various toxin serotypes. Detailed analyses of flagellin from two strains of C. botulinum demonstrated that the protein is modified by a novel glycan moiety of mass 417 Da in O-linkage. Bioinformatic analysis of available C. botulinum genomes identified a flagellar glycosylation island containing homologs of genes recently identified in Campylobacter coli that have been shown to be responsible for the biosynthesis of legionaminic acid derivatives. Structural characterization of the carbohydrate moiety was completed utilizing both MS and NMR spectroscopy, and it was shown to be a novel legionaminic acid derivative, 7-acetamido-5-(N-methyl-glutam-4-yl)-amino-3,5,7,9-tetradeoxy-D-glycero-alpha-D-galacto-nonulosonic acid, (alphaLeg5GluNMe7Ac). Electron transfer dissociation MS with and without collision-activated dissociation was utilized to map seven sites of O-linked glycosylation, eliminating the need for chemical derivatization of tryptic peptides prior to analysis. Marker ions for novel glycans, as well as a unique C-terminal flagellin peptide marker ion, were identified in a top-down analysis of the intact protein. These ions have the potential for use in for rapid detection and discrimination of C. botulinum cells, indicating botulinum neurotoxin contamination. This is the first report of glycosylation of Gram-positive flagellar proteins by the 'sialic acid-like' nonulosonate sugar, legionaminic acid.

Recombinant, structually unique Kazal-type proteinase inhibitor retains activity when C-terminally extended and glycosylated

Czech Academy of Sciences Publication Activity Database

Kludkiewicz, B.; Kodrík, Dalibor; Grzelak, K.; Nirmala, X.; Sehnal, František

2005-01-01

Roč. 43, č. 2 (2005), s. 94-102 ISSN 1046-5928 R&D Projects: GA AV ČR(CZ) IBS5007316 Grant - others:Centre of Excellence in Molecular Biology at the Institute of Biochemistry and Biophysics(BE) ICA-CT-2000-700010; NATO(BE) LST.CLG.979223 Institutional research plan: CEZ:AV0Z50070508 Keywords : fusion proteins * glycosylation * Kazal domain Subject RIV: CE - Biochemistry Impact factor: 1.553, year: 2005

Defining the phenotype and diagnostic considerations in adults with congenital disorders of N-linked glycosylation

NARCIS (Netherlands)

Wolthuis, D.F.; Janssen, M.C.H.; Cassiman, D.; Lefeber, D.J.; Morava-Kozicz, E.

2014-01-01

Congenital disorders of N-glycosylation (CDG) form a rapidly growing group of more than 20 inborn errors of metabolism. Most patients are identified at the pediatric age with multisystem disease. There is no systematic review on the long-term outcome and clinical presentation in adult patients.

In-vivo biological activity and glycosylation analysis of a biosimilar recombinant human follicle-stimulating hormone product (Bemfola compared with its reference medicinal product (GONAL-f.

Directory of Open Access Journals (Sweden)

Renato Mastrangeli

Full Text Available Recombinant human follicle-stimulating hormone (r-hFSH is widely used in fertility treatment. Although biosimilar versions of r-hFSH (follitropin alfa are currently on the market, given their structural complexity and manufacturing process, it is important to thoroughly evaluate them in comparison with the reference product. This evaluation should focus on how they differ (e.g., active component molecular characteristics, impurities and potency, as this could be associated with clinical outcome. This study compared the site-specific glycosylation profile and batch-to-batch variability of the in-vivo bioactivity of Bemfola, a biosimilar follitropin alfa, with its reference medicinal product GONAL-f. The focus of this analysis was the site-specific glycosylation at asparagine (Asn 52 of the α-subunit of FSH, owing to the pivotal role of Asn52 glycosylation in FSH receptor (FSHR activation/signalling. Overall, Bemfola had bulkier glycan structures and greater sialylation than GONAL-f. The nominal specific activity for both Bemfola and GONAL-f is 13,636 IU/mg. Taking into account both the determined potency and the nominal amount the average specific activity of Bemfola was 14,522 IU/mg (105.6% of the nominal value, which was greater than the average specific activity observed for GONAL-f (13,159 IU/mg; 97.3% of the nominal value; p = 0.0048, although this was within the range stated in the product label. A higher batch-to-batch variability was also observed for Bemfola versus GONAL-f (coefficient of variation: 8.3% vs 5.8%. A different glycan profile was observed at Asn52 in Bemfola compared with GONAL-f (a lower proportion of bi-antennary structures [~53% vs ~77%], and a higher proportion of tri-antennary [~41% vs ~23%] and tetra-antennary structures [~5% vs <1%]. These differences in the Asn52 glycan profile might potentially lead to differences in FSHR activation. This, together with the greater bioactivity and higher batch-to-batch variability

Modeling the mechanism of glycosylation reactions between ethanol, 1,2-ethanediol and methoxymethanol.

Science.gov (United States)

Azofra, Luis Miguel; Alkorta, Ibon; Toro-Labbé, Alejandro; Elguero, José

2013-09-07

The mechanism of the S(N)2 model glycosylation reaction between ethanol, 1,2-ethanediol and methoxymethanol has been studied theoretically at the B3LYP/6-311+G(d,p) computational level. Three different types of reactions have been explored: (i) the exchange of hydroxyl groups between these model systems; (ii) the basic catalysis reactions by combination of the substrates as glycosyl donors (neutral species) and acceptors (enolate species); and (iii) the effect on the reaction profile of an explicit H2O molecule in the reactions considered in (ii). The reaction force, the electronic chemical potential and the reaction electronic flux have been characterized for the reaction path in each case. Energy calculations show that methoxymethanol is the worst glycosyl donor model among the ones studied here, while 1,2-ethanediol is the best, having the lowest activation barrier of 74.7 kJ mol(-1) for the reaction between this one and the ethanolate as the glycosyl acceptor model. In general, the presence of direct interactions between the atoms involved in the penta-coordinated TS increases the activation energies of the processes.

Mapping Sites of O-Glycosylation and Fringe Elongation on Drosophila Notch*

Science.gov (United States)

Harvey, Beth M.; Rana, Nadia A.; Moss, Hillary; Leonardi, Jessica; Jafar-Nejad, Hamed; Haltiwanger, Robert S.

2016-01-01

Glycosylation of the Notch receptor is essential for its activity and serves as an important modulator of signaling. Three major forms of O-glycosylation are predicted to occur at consensus sites within the epidermal growth factor-like repeats in the extracellular domain of the receptor: O-fucosylation, O-glucosylation, and O-GlcNAcylation. We have performed comprehensive mass spectral analyses of these three types of O-glycosylation on Drosophila Notch produced in S2 cells and identified peptides containing all 22 predicted O-fucose sites, all 18 predicted O-glucose sites, and all 18 putative O-GlcNAc sites. Using semiquantitative mass spectral methods, we have evaluated the occupancy and relative amounts of glycans at each site. The majority of the O-fucose sites were modified to high stoichiometries. Upon expression of the β3-N-acetylglucosaminyltransferase Fringe with Notch, we observed varying degrees of elongation beyond O-fucose monosaccharide, indicating that Fringe preferentially modifies certain sites more than others. Rumi modified O-glucose sites to high stoichiometries, although elongation of the O-glucose was site-specific. Although the current putative consensus sequence for O-GlcNAcylation predicts 18 O-GlcNAc sites on Notch, we only observed apparent O-GlcNAc modification at five sites. In addition, we performed mass spectral analysis on endogenous Notch purified from Drosophila embryos and found that the glycosylation states were similar to those found on Notch from S2 cells. These data provide foundational information for future studies investigating the mechanisms of how O-glycosylation regulates Notch activity. PMID:27268051

Toward stable genetic engineering of human o-glycosylation in plants

DEFF Research Database (Denmark)

Yang, Zhang; Bennett, Eric Paul; Jørgensen, Bodil

2012-01-01

Glycosylation is the most abundant and complex posttranslational modification to be considered for recombinant production of therapeutic proteins. Mucin-type (N-acetylgalactosamine [GalNAc]-type) O-glycosylation is found in eumetazoan cells but absent in plants and yeast, making these cell types...... an obvious choice for de novo engineering of this O-glycosylation pathway. We previously showed that transient implementation of O-glycosylation capacity in plants requires introduction of the synthesis of the donor substrate UDP-GalNAc and one or more polypeptide GalNAc-transferases for incorporating Gal......NAc residues into proteins. Here, we have stably engineered O-glycosylation capacity in two plant cell systems, soil-grown Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum) Bright Yellow-2 suspension culture cells. Efficient GalNAc O-glycosylation of two stably coexpressed substrate O...

N-glycosylated catalytic unit meets O-glycosylated propeptide: complex protein architecture in a fungal hexosaminidase

Czech Academy of Sciences Publication Activity Database

Plíhal, Ondřej; Sklenář, Jan; Kmoníčková, J.; Man, Petr; Pompach, Petr; Havlíček, Vladimír; Křen, Vladimír; Bezouška, Karel

2004-01-01

Roč. 32, č. 5 (2004), s. 764-765 ISSN 0300-5127 R&D Projects: GA ČR GA203/04/1045 Institutional research plan: CEZ:AV0Z5020903 Keywords : asperillus oryzoe * glycosyl hydrolase * preproprotein Subject RIV: EE - Microbiology, Virology Impact factor: 2.267, year: 2004

Processing, fusogenicity, virion incorporation and CXCR4-binding activity of a feline immunodeficiency virus envelope glycoprotein lacking the two conserved N-glycosylation sites at the C-terminus of the V3 domain.

Science.gov (United States)

González, Silvia A; Affranchino, José L

2016-07-01

The process of feline immunodeficiency virus (FIV) entry into its target cells is initiated by the association of the surface (SU) subunit of the viral envelope glycoprotein (Env) with the cellular receptors CD134 and CXCR4. This event is followed by the fusion of the viral and cellular membranes, which is mediated by the transmembrane (TM) subunit of Env. We and others have previously demonstrated that the V3 domain of the SU subunit of Env is essential for CXCR4 binding. Of note, there are two contiguous and highly conserved potential N-glycosylation sites ((418)NST(420) and (422)NLT(424)) located at the C-terminal side of the V3 domain. We therefore decided to study the relevance for Env functions of these N-glycosylation motifs and found that disruption of both of them by introducing the N418Q/N422Q double amino acid substitution drastically impairs Env processing into the SU and TM subunits. Moreover, the simultaneous mutation of these N-glycosylation sites prevents Env incorporation into virions and Env-mediated cell-to-cell fusion. Notably, a recombinant soluble version of the SU glycoprotein carrying the double amino acid replacement N418Q/N422Q at the V3 C-terminal side binds to CXCR4 with an efficiency similar to that of wild-type SU.

Mutations in four glycosyl hydrolases reveal a highly coordinated pathway for rhodopsin biosynthesis and N-glycan trimming in Drosophila melanogaster.

Directory of Open Access Journals (Sweden)

Erica E Rosenbaum

2014-05-01

Full Text Available As newly synthesized glycoproteins move through the secretory pathway, the asparagine-linked glycan (N-glycan undergoes extensive modifications involving the sequential removal and addition of sugar residues. These modifications are critical for the proper assembly, quality control and transport of glycoproteins during biosynthesis. The importance of N-glycosylation is illustrated by a growing list of diseases that result from defects in the biosynthesis and processing of N-linked glycans. The major rhodopsin in Drosophila melanogaster photoreceptors, Rh1, is highly unique among glycoproteins, as the N-glycan appears to be completely removed during Rh1 biosynthesis and maturation. However, much of the deglycosylation pathway for Rh1 remains unknown. To elucidate the key steps in Rh1 deglycosylation in vivo, we characterized mutant alleles of four Drosophila glycosyl hydrolases, namely α-mannosidase-II (α-Man-II, α-mannosidase-IIb (α-Man-IIb, a β-N-acetylglucosaminidase called fused lobes (Fdl, and hexosaminidase 1 (Hexo1. We have demonstrated that these four enzymes play essential and unique roles in a highly coordinated pathway for oligosaccharide trimming during Rh1 biosynthesis. Our results reveal that α-Man-II and α-Man-IIb are not isozymes like their mammalian counterparts, but rather function at distinct stages in Rh1 maturation. Also of significance, our results indicate that Hexo1 has a biosynthetic role in N-glycan processing during Rh1 maturation. This is unexpected given that in humans, the hexosaminidases are typically lysosomal enzymes involved in N-glycan catabolism with no known roles in protein biosynthesis. Here, we present a genetic dissection of glycoprotein processing in Drosophila and unveil key steps in N-glycan trimming during Rh1 biosynthesis. Taken together, our results provide fundamental advances towards understanding the complex and highly regulated pathway of N-glycosylation in vivo and reveal novel insights

Mutations in Four Glycosyl Hydrolases Reveal a Highly Coordinated Pathway for Rhodopsin Biosynthesis and N-Glycan Trimming in Drosophila melanogaster

Science.gov (United States)

Rosenbaum, Erica E.; Vasiljevic, Eva; Brehm, Kimberley S.; Colley, Nansi Jo

2014-01-01

As newly synthesized glycoproteins move through the secretory pathway, the asparagine-linked glycan (N-glycan) undergoes extensive modifications involving the sequential removal and addition of sugar residues. These modifications are critical for the proper assembly, quality control and transport of glycoproteins during biosynthesis. The importance of N-glycosylation is illustrated by a growing list of diseases that result from defects in the biosynthesis and processing of N-linked glycans. The major rhodopsin in Drosophila melanogaster photoreceptors, Rh1, is highly unique among glycoproteins, as the N-glycan appears to be completely removed during Rh1 biosynthesis and maturation. However, much of the deglycosylation pathway for Rh1 remains unknown. To elucidate the key steps in Rh1 deglycosylation in vivo, we characterized mutant alleles of four Drosophila glycosyl hydrolases, namely α-mannosidase-II (α-Man-II), α-mannosidase-IIb (α-Man-IIb), a β-N-acetylglucosaminidase called fused lobes (Fdl), and hexosaminidase 1 (Hexo1). We have demonstrated that these four enzymes play essential and unique roles in a highly coordinated pathway for oligosaccharide trimming during Rh1 biosynthesis. Our results reveal that α-Man-II and α-Man-IIb are not isozymes like their mammalian counterparts, but rather function at distinct stages in Rh1 maturation. Also of significance, our results indicate that Hexo1 has a biosynthetic role in N-glycan processing during Rh1 maturation. This is unexpected given that in humans, the hexosaminidases are typically lysosomal enzymes involved in N-glycan catabolism with no known roles in protein biosynthesis. Here, we present a genetic dissection of glycoprotein processing in Drosophila and unveil key steps in N-glycan trimming during Rh1 biosynthesis. Taken together, our results provide fundamental advances towards understanding the complex and highly regulated pathway of N-glycosylation in vivo and reveal novel insights into the

Prion Propagation in Cells Expressing PrP Glycosylation Mutants ▿

Science.gov (United States)

Salamat, Muhammad K.; Dron, Michel; Chapuis, Jérôme; Langevin, Christelle; Laude, Hubert

2011-01-01

Infection by prions involves conversion of a host-encoded cell surface protein (PrPC) to a disease-related isoform (PrPSc). PrPC carries two glycosylation sites variably occupied by complex N-glycans, which have been suggested by previous studies to influence the susceptibility to these diseases and to determine characteristics of prion strains. We used the Rov cell system, which is susceptible to sheep prions, to generate a series of PrPC glycosylation mutants with mutations at one or both attachment sites. We examined their subcellular trafficking and ability to convert into PrPSc and to sustain stable prion propagation in the absence of wild-type PrP. The susceptibility to infection of mutants monoglycosylated at either site differed dramatically depending on the amino acid substitution. Aglycosylated double mutants showed overaccumulation in the Golgi compartment and failed to be infected. Introduction of an ectopic glycosylation site near the N terminus fully restored cell surface expression of PrP but not convertibility into PrPSc, while PrPC with three glycosylation sites conferred cell permissiveness to infection similarly to the wild type. In contrast, predominantly aglycosylated molecules with nonmutated N-glycosylation sequons, produced in cells expressing glycosylphosphatidylinositol-anchorless PrPC, were able to form infectious PrPSc. Together our findings suggest that glycosylation is important for efficient trafficking of anchored PrP to the cell surface and sustained prion propagation. However, properly trafficked glycosylation mutants were not necessarily prone to conversion, thus making it difficult in such studies to discern whether the amino acid changes or glycan chain removal most influences the permissiveness to prion infection. PMID:21248032

N-O linkage in carbohydrates and glycoconjugates.

Science.gov (United States)

Chen, N; Xie, J

2016-11-29

The importance of oligosaccharides and their conjugates in various biological and pathological processes has stimulated growing interest in the development of (neo)glycoconjugates. Thanks to its high nucleophilicity, hydroxylamine has been employed as a powerful chemoselective ligation tool. Great effort has been focused on carbohydrates bearing aminooxy or N-hydroxy amino groups for organic synthesis, glycobiology and drug discovery. This review provides an overview of N-O linked carbohydrates and glycoconjugates, focusing particularly on the synthetic methodologies and chemical and physicochemical properties as well as biological and medical applications of N-glycosyl and O-glycosyl hydroxylamines, N-hydroxy amino and O-amino sugar as well as sugar aminooxy acid derivatives.

Glycosylation of Hemagglutinin and Neuraminidase of Influenza A Virus as Signature for Ecological Spillover and Adaptation among Influenza Reservoirs

Directory of Open Access Journals (Sweden)

Paul Kim

2018-04-01

Full Text Available Glycosylation of the hemagglutinin (HA and neuraminidase (NA of the influenza provides crucial means for immune evasion and viral fitness in a host population. However, the time-dependent dynamics of each glycosylation sites have not been addressed. We monitored the potential N-linked glycosylation (NLG sites of over 10,000 HA and NA of H1N1 subtype isolated from human, avian, and swine species over the past century. The results show a shift in glycosylation sites as a hallmark of 1918 and 2009 pandemics, and also for the 1976 “abortive pandemic”. Co-segregation of particular glycosylation sites was identified as a characteristic of zoonotic transmission from animal reservoirs, and interestingly, of “reverse zoonosis” of human viruses into swine populations as well. After the 2009 pandemic, recent isolates accrued glycosylation at canonical sites in HA, reflecting gradual seasonal adaptation, and a novel glycosylation in NA as an independent signature for adaptation among humans. Structural predictions indicated a remarkably pleiotropic influence of glycans on multiple HA epitopes for immune evasion, without sacrificing the receptor binding of HA or the activity of NA. The results provided the rationale for establishing the ecological niche of influenza viruses among the reservoir and could be implemented for influenza surveillance and improving pandemic preparedness.

Glycosylation of Hemagglutinin and Neuraminidase of Influenza A Virus as Signature for Ecological Spillover and Adaptation among Influenza Reservoirs

Science.gov (United States)

Kim, Paul; Jang, Yo Han; Kwon, Soon Bin; Lee, Chung Min; Han, Gyoonhee; Seong, Baik Lin

2018-01-01

Glycosylation of the hemagglutinin (HA) and neuraminidase (NA) of the influenza provides crucial means for immune evasion and viral fitness in a host population. However, the time-dependent dynamics of each glycosylation sites have not been addressed. We monitored the potential N-linked glycosylation (NLG) sites of over 10,000 HA and NA of H1N1 subtype isolated from human, avian, and swine species over the past century. The results show a shift in glycosylation sites as a hallmark of 1918 and 2009 pandemics, and also for the 1976 “abortive pandemic”. Co-segregation of particular glycosylation sites was identified as a characteristic of zoonotic transmission from animal reservoirs, and interestingly, of “reverse zoonosis” of human viruses into swine populations as well. After the 2009 pandemic, recent isolates accrued glycosylation at canonical sites in HA, reflecting gradual seasonal adaptation, and a novel glycosylation in NA as an independent signature for adaptation among humans. Structural predictions indicated a remarkably pleiotropic influence of glycans on multiple HA epitopes for immune evasion, without sacrificing the receptor binding of HA or the activity of NA. The results provided the rationale for establishing the ecological niche of influenza viruses among the reservoir and could be implemented for influenza surveillance and improving pandemic preparedness. PMID:29642453

[Non-enzymatic glycosylation of dietary protein in vitro].

Science.gov (United States)

Bednykh, B S; Evdokimov, I A; Sokolov, A I

2015-01-01

Non-enzymatic glycosylation of proteins, based on discovered by Mayarn reaction of carbohydrate aldehyde group with a free amino group of a protein molecule, is well known to experts in biochemistry of food industry. Generated brown solid in some cases give the product marketable qualities--crackling bread--in others conversely, worsen the product. The biological effects of far-advanced products of non-enzymatic protein glycosylation reaction have not been studied enough, although it was reported previously that they are not split by digestive enzymes and couldn't be absorbed by animals. The objective of this work was to compare the depth of glycosylation of different food proteins of animal and vegetable origin. The objects of the study were proteins of animal (casein, lactoglobulin, albumin) and vegetable (soy isolate, proteins of rice flour, buckwheat, oatmeal) origin, glucose and fructose were selected as glycosylation agents, exposure 15 days at 37 degrees C. Lactoglobulin was glycosylated to a lesser extent among the proteins of animal origin while protein of oatmeal was glycosylated in the least degree among vegetable proteins. Conversely, such proteins as casein and soya isolate protein bound rather large amounts of carbohydrates. Fructose binding with protein was generally higher than the binding of glucose. The only exception was a protein of oatmeal. When of glucose and fructose simultaneously presented in the incubation medium, glucose binding usually increased while binding of fructose, in contrast, reduced. According to the total amount of carbohydrate (mcg), which is able to attach a protein (mg) the studied food proteins located in the following order: albumin (38) > soy protein isolate (23) > casein (15,) > whey protein rice flour protein (6) > protein from buckwheat flour (3) > globulin (2) > protein of oatmeal (0.3). The results obtained are to be used to select the optimal combination of proteins and carbohydrates, in which the glycosylation

Characterization of kallikrein-related peptidase 4 glycosylations.

Science.gov (United States)

Yamakoshi, Yasuo; Yamakoshi, Fumiko; Hu, Jan C-C; Simmer, James P

2011-12-01

Kallikrein-related peptidase 4 (KLK4) is a glycosylated serine protease that functions in the maturation (hardening) of dental enamel. Pig and mouse KLK4 contain three potential N-glycosylation sites. We isolated KLK4 from developing pig and mouse molars and characterized their N-glycosylations. N-glycans were enzymatically released by digestion with N-glycosidase F and fluorescently labeled with 2-aminobenzoic acid. Normal-phase high-performance liquid chromatography (NP-HPLC) revealed N-glycans with no, or with one, two, or three sialic acid attachments in pig KLK4 and with no, or with one or two sialic acid attachments in mouse KLK4. The labeled N-glycans were digested with sialidase to generate the asialo N-glycan cores that were fractionated by reverse-phase HPLC, and their retention times were compared with similarly labeled glycan standards. The purified cores were characterized by mass spectrometric and monosaccharide composition analyses. We determined that pig and mouse KLK4 have NA2 and NA2F biantennary N-glycan cores. The pig triantennary core is NA3. The mouse triantennary core is NA3 with a fucose connected by an α1-6 linkage, indicating that it is attached to the first N-acetyglucosamine (NA3F). We conclude that pig KLK4 has NA2, NA2F, and NA3 N-glycan cores with no, or with one, two, or three sialic acids. Mouse KLK4 has NA2, NA2F, and NA3F N-glycan cores with no, or with one or two sialic acids. © 2011 Eur J Oral Sci.

N-glycosylation of colorectal cancer tissues: a liquid chromatography and mass spectrometry-based investigation.

Science.gov (United States)

Balog, Crina I A; Stavenhagen, Kathrin; Fung, Wesley L J; Koeleman, Carolien A; McDonnell, Liam A; Verhoeven, Aswin; Mesker, Wilma E; Tollenaar, Rob A E M; Deelder, André M; Wuhrer, Manfred

2012-09-01

Colorectal cancer is the third most common cancer worldwide with an annual incidence of ~1 million cases and an annual mortality rate of ~655,000 individuals. There is an urgent need for identifying novel targets to develop more sensitive, reliable, and specific tests for early stage detection of colon cancer. Post-translational modifications are known to play an important role in cancer progression and immune surveillance of tumors. In the present study, we compared the N-glycan profiles from 13 colorectal cancer tumor tissues and corresponding control colon tissues. The N-glycans were enzymatically released, purified, and labeled with 2-aminobenzoic acid. Aliquots were profiled by hydrophilic interaction liquid chromatography (HILIC-HPLC) with fluorescence detection and by negative mode MALDI-TOF-MS. Using partial least squares discriminant analysis to investigate the N-glycosylation changes in colorectal cancer, an excellent separation and prediction ability were observed for both HILIC-HPLC and MALDI-TOF-MS data. For structure elucidation, information from positive mode ESI-ion trap-MS/MS and negative mode MALDI-TOF/TOF-MS was combined. Among the features with a high separation power, structures containing a bisecting GlcNAc were found to be decreased in the tumor, whereas sulfated glycans, paucimannosidic glycans, and glycans containing a sialylated Lewis type epitope were shown to be increased in tumor tissues. In addition, core-fucosylated high mannose N-glycans were detected in tumor samples. In conclusion, the combination of HILIC and MALDI-TOF-MS profiling of N-glycans with multivariate statistical analysis demonstrated its potential for identifying N-glycosylation changes in colorectal cancer tissues and provided new leads that might be used as candidate biomarkers.

Comparative Glycoproteome Analysis: Dynamics of Protein Glycosylation during Metamorphic Transition from Pelagic to Benthic Life Stages in Three Invertebrates

KAUST Repository

Chandramouli, Kondethimmanahalli

2012-02-03

The life cycle of most benthic marine invertebrates has two distinct stages: the pelagic larval stage and the sessile juvenile stage. The transition between the larval stage and the juvenile stage is often abrupt and may be triggered by post-translational modification of proteins. Glycosylation, a very important post-translational modification, influences the biological activity of proteins. We used two-dimensional gel electrophoresis (2-DE) followed by glycoprotein-specific fluorescence staining and mass spectrometry with the goal of identifying glycosylation pattern changes during larval settlement and metamorphosis in barnacles, bryozoans, and polychaetes. Our results revealed substantial changes in the protein glycosylation patterns from larval to juvenile stages. Before metamorphosis, the degree of protein glycosylation was high in the barnacle Balanus (=Amphibalanus) amphitrite and the spionid polychaete Pseudopolydora vexillosa, whereas it increased after metamorphosis in the bryozoan Bugula neritina. We identified 19 abundant and differentially glycosylated proteins in these three species. Among the proteins, cellular stress- and metabolism-related proteins exhibited distinct glycosylation in B. amphitrite and B. neritina, whereas fatty acid metabolism-related proteins were abundantly glycosylated in P. vexillosa. Furthermore, the protein and gene expression analysis of some selected glycoproteins revealed that the degree of protein glycosylation did not always complement with transcriptional and translational changes associated with the larval-juvenile transition. The current study provides preliminary information on protein glycosylation in marine invertebrates that will serve as a solid basis for future comprehensive analysis of glycobiology during larval settlement and metamorphosis. © 2011 American Chemical Society.

Genome-scale metabolic model of Pichia pastoris with native and humanized glycosylation of recombinant proteins.

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Irani, Zahra Azimzadeh; Kerkhoven, Eduard J; Shojaosadati, Seyed Abbas; Nielsen, Jens

2016-05-01

Pichia pastoris is used for commercial production of human therapeutic proteins, and genome-scale models of P. pastoris metabolism have been generated in the past to study the metabolism and associated protein production by this yeast. A major challenge with clinical usage of recombinant proteins produced by P. pastoris is the difference in N-glycosylation of proteins produced by humans and this yeast. However, through metabolic engineering, a P. pastoris strain capable of producing humanized N-glycosylated proteins was constructed. The current genome-scale models of P. pastoris do not address native nor humanized N-glycosylation, and we therefore developed ihGlycopastoris, an extension to the iLC915 model with both native and humanized N-glycosylation for recombinant protein production, but also an estimation of N-glycosylation of P. pastoris native proteins. This new model gives a better prediction of protein yield, demonstrates the effect of the different types of N-glycosylation of protein yield, and can be used to predict potential targets for strain improvement. The model represents a step towards a more complete description of protein production in P. pastoris, which is required for using these models to understand and optimize protein production processes. © 2015 Wiley Periodicals, Inc.

SnapShot: O-Glycosylation Pathways across Kingdoms

DEFF Research Database (Denmark)

Joshi, Hiren J.; Narimatsu, Yoshiki; Schjoldager, Katrine T.

2018-01-01

O-glycosylation is one of the most abundant and diverse types of post-translational modifications of proteins. O-glycans modulate the structure, stability, and function of proteins and serve generalized as well as highly specific roles in most biological processes. This ShapShot presents types of......-glycans found in different organisms and their principle biosynthetic pathways...

Enhanced SCAP glycosylation by inflammation induces macrophage foam cell formation.

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Chao Zhou

Full Text Available Inflammatory stress promotes foam cell formation by disrupting LDL receptor feedback regulation in macrophages. Sterol Regulatory Element Binding Proteins (SREBPs Cleavage-Activating Protein (SCAP glycosylation plays crucial roles in regulating LDL receptor and 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCoAR feedback regulation. The present study was to investigate if inflammatory stress disrupts LDL receptor and HMGCoAR feedback regulation by affecting SCAP glycosylation in THP-1 macrophages. Intracellular cholesterol content was assessed by Oil Red O staining and quantitative assay. The expression of molecules controlling cholesterol homeostasis was examined using real-time quantitative RT-PCR and Western blotting. The translocation of SCAP from the endoplasmic reticulum (ER to the Golgi was detected by confocal microscopy. We demonstrated that exposure to inflammatory cytokines increased lipid accumulation in THP-1 macrophages, accompanying with an increased SCAP expression even in the presence of a high concentration of LDL. These inflammatory cytokines also prolonged the half-life of SCAP by enhancing glycosylation of SCAP due to the elevated expression of the Golgi mannosidase II. This may enhance translocation and recycling of SCAP between the ER and the Golgi, escorting more SREBP2 from the ER to the Golgi for activation by proteolytic cleavages as evidenced by an increased N-terminal of SREBP2 (active form. As a consequence, the LDL receptor and HMGCoAR expression were up-regulated. Interestingly, these effects could be blocked by inhibitors of Golgi mannosidases. Our results indicated that inflammation increased native LDL uptake and endogenous cholesterol de novo synthesis, thereby causing foam cell formation via increasing transcription and protein glycosylation of SCAP in macrophages. These data imply that inhibitors of Golgi processing enzymes might have a potential vascular-protective role in prevention of atherosclerotic foam

Plant glyco-biotechnology on the way to synthetic biology

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Andreas eLoos

2014-10-01

Full Text Available Plants are increasingly being used for the production of recombinant proteins. One reason is that plants are highly amenable for glycan engineering processes and allow the production of therapeutic proteins with increased efficacies due to optimized glycosylation profiles. Removal and insertion of glycosylation reactions by knock-out/knock-down approaches and introduction of glycosylation enzymes have paved the way for the humanization of the plant glycosylation pathway. The insertion of heterologous enzymes at exactly the right stage of the existing glycosylation pathway has turned out to be of utmost importance for optimal results. To enable such precise targeting chimeric enzymes have been constructed. In this short review we will exemplify the importance of correct targeting of glycosyltransferases, we will give an overview of the targeting mechanism of glycosyltransferases, describe chimeric enzymes used in plant N-glycosylation engineering and illustrate how plant glycoengineering builds on the tools offered by synthetic biology to construct such chimeric enzymes.

Comparative Analysis of Whey N-Glycoproteins in Human Colostrum and Mature Milk Using Quantitative Glycoproteomics.

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Cao, Xueyan; Song, Dahe; Yang, Mei; Yang, Ning; Ye, Qing; Tao, Dongbing; Liu, Biao; Wu, Rina; Yue, Xiqing

2017-11-29

Glycosylation is a ubiquitous post-translational protein modification that plays a substantial role in various processes. However, whey glycoproteins in human milk have not been completely profiled. Herein, we used quantitative glycoproteomics to quantify whey N-glycosylation sites and their alteration in human milk during lactation; 110 N-glycosylation sites on 63 proteins and 91 N-glycosylation sites on 53 proteins were quantified in colostrum and mature milk whey, respectively. Among these, 68 glycosylation sites on 38 proteins were differentially expressed in human colostrum and mature milk whey. These differentially expressed N-glycoproteins were highly enriched in "localization", "extracellular region part", and "modified amino acid binding" according to gene ontology annotation and mainly involved in complement and coagulation cascades pathway. These results shed light on the glycosylation sites, composition and biological functions of whey N-glycoproteins in human colostrum and mature milk, and provide substantial insight into the role of protein glycosylation during infant development.

Synthesis and biological activity of new homolupanes and homolupane saponins

Czech Academy of Sciences Publication Activity Database

Sidoryk, K.; Korda, A.; Rárová, Lucie; Oklešťková, Jana; Strnad, Miroslav; Cmoch, P.; Pakulski, Z.; Gwardiak, K.; Karczewski, R.; Luboradzki, R.

Roč. 71, č. 13 ( 2015 ), s. 2004-2012 ISSN 0040-4020 R&D Projects: GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : Homobetulin * Homobetulinic acid * Glycosylation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.645, year: 2015

N-Glycosylation instead of cholesterol mediates oligomerization and apical sorting of GPI-APs in FRT cells.

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Imjeti, Naga Salaija; Lebreton, Stéphanie; Paladino, Simona; de la Fuente, Erwin; Gonzalez, Alfonso; Zurzolo, Chiara

2011-12-01

Sorting of glycosylphosphatidyl-inositol--anchored proteins (GPI-APs) in polarized epithelial cells is not fully understood. Oligomerization in the Golgi complex has emerged as the crucial event driving apical segregation of GPI-APs in two different kind of epithelial cells, Madin-Darby canine kidney (MDCK) and Fisher rat thyroid (FRT) cells, but whether the mechanism is conserved is unknown. In MDCK cells cholesterol promotes GPI-AP oligomerization, as well as apical sorting of GPI-APs. Here we show that FRT cells lack this cholesterol-driven oligomerization as apical sorting mechanism. In these cells both apical and basolateral GPI-APs display restricted diffusion in the Golgi likely due to a cholesterol-enriched membrane environment. It is striking that N-glycosylation is the critical event for oligomerization and apical sorting of GPI-APs in FRT cells but not in MDCK cells. Our data indicate that at least two mechanisms exist to determine oligomerization in the Golgi leading to apical sorting of GPI-APs. One depends on cholesterol, and the other depends on N-glycosylation and is insensitive to cholesterol addition or depletion.

Involvement of Aberrant Glycosylation in Thyroid Cancer

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Eiji Miyoshi

2010-01-01

Full Text Available Glycosylation is one of the most common posttranslational modification reactions and nearly half of all known proteins in eukaryotes are glycosylated. In fact, changes in oligosaccharides structures are associated with many physiological and pathological events, including cell growth, migration and differentiation, and tumor invasion. Therefore, functional glycomics, which is a comprehensive study of the structures and functions of glycans, is attracting the increasing attention of scientists in various fields of life science. In cases of thyroid cancer, the biological characters and prognosis are completely different in each type of histopathology, and their oligosaccharide structures as well as the expression of glycosyltransferases are also different. In this review, we summarized our previous papers on oligosaccharides and thyroid cancers and discussed a possible function of oligosaccharides in the carcinogenesis in thyroid cancer.

The effects of marine carbohydrates and glycosylated compounds on human health.

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Kang, Hee-Kyoung; Seo, Chang Ho; Park, Yoonkyung

2015-03-16

Marine organisms have been recognized as a valuable source of bioactive compounds with industrial and nutraceutical potential. Recently, marine-derived carbohydrates, including polysaccharides and low molecular weight glycosylated oligosaccharides, have attracted much attention because of their numerous health benefits. Moreover, several studies have reported that marine carbohydrates exhibit various biological activities, including antioxidant, anti-infection, anticoagulant, anti-inflammatory, and anti-diabetic effects. The present review discusses the potential industrial applications of bioactive marine carbohydrates for health maintenance and disease prevention. Furthermore, the use of marine carbohydrates in food, cosmetics, agriculture, and environmental protection is discussed.

Carbohydrates on Proteins: Site-Specific Glycosylation Analysis by Mass Spectrometry

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Zhu, Zhikai; Desaire, Heather

2015-07-01

Glycosylation on proteins adds complexity and versatility to these biologically vital macromolecules. To unveil the structure-function relationship of glycoproteins, glycopeptide-centric analysis using mass spectrometry (MS) has become a method of choice because the glycan is preserved on the glycosylation site and site-specific glycosylation profiles of proteins can be readily determined. However, glycopeptide analysis is still challenging given that glycopeptides are usually low in abundance and relatively difficult to detect and the resulting data require expertise to analyze. Viewing the urgent need to address these challenges, emerging methods and techniques are being developed with the goal of analyzing glycopeptides in a sensitive, comprehensive, and high-throughput manner. In this review, we discuss recent advances in glycoprotein and glycopeptide analysis, with topics covering sample preparation, analytical separation, MS and tandem MS techniques, as well as data interpretation and automation.

Antibody glycosylation and its impact on the pharmacokinetics and pharmacodynamics of monoclonal antibodies and Fc-fusion proteins.

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Liu, Liming

2015-06-01

Understanding the impact of glycosylation and keeping a close control on glycosylation of product candidates are required for both novel and biosimilar monoclonal antibodies (mAbs) and Fc-fusion protein development to ensure proper safety and efficacy profiles. Most therapeutic mAbs are of IgG class and contain a glycosylation site in the Fc region at amino acid position 297 and, in some cases, in the Fab region. For Fc-fusion proteins, glycosylation also frequently occurs in the fusion partners. Depending on the expression host, glycosylation patterns in mAb or Fc-fusions can be significantly different, thus significantly impacting the pharmacokinetics (PK) and pharmacodynamics (PD) of mAbs. Glycans that have a major impact on PK and PD of mAb or Fc-fusion proteins include mannose, sialic acids, fucose (Fuc), and galactose (Gal). Mannosylated glycans can impact the PK of the molecule, leading to reduced exposure and potentially lower efficacy. The level of sialic acid, N-acetylneuraminic acid (NANA), can also have a significant impact on the PK of Fc-fusion molecules. Core Fuc in the glycan structure reduces IgG antibody binding to IgG Fc receptor IIIa relative to IgG lacking Fuc, resulting in decreased antibody-dependent cell-mediated cytotoxicity (ADCC) activities. Glycoengineered Chinese hamster ovary (CHO) expression systems can produce afucosylated mAbs that have increased ADCC activities. Terminal Gal in a mAb is important in the complement-dependent cytotoxicity (CDC) in that lower levels of Gal reduce CDC activity. Glycans can also have impacts on the safety of mAb. mAbs produced in murine myeloma cells such as NS0 and SP2/0 contain glycans such as Galα1-3Galβ1-4N-acetylglucosamine-R and N-glycolylneuraminic acid (NGNA) that are not naturally present in humans and can be immunogenic when used as therapeutics. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

HEK293T cell lines defective for O-linked glycosylation.

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James M Termini

Full Text Available Here we describe derivatives of the HEK293T cell line that are defective in their ability to generate mucin-type O-linked glycosylation. Using CRISPR/Cas9 and a single-cell GFP-sorting procedure, the UDP-galactose-4-epimerase (GALE, galactokinase 1 (GALK1, and galactokinase 2 (GALK2 genes were knocked out individually and in combinations with greater than 90% of recovered clones having the desired mutations. Although HEK293T cells are tetraploid, we found this approach to be an efficient method to target and disrupt all 4 copies of the target gene. Deficient glycosylation in the GALE knockout cell line could be rescued by the addition of galactose and N-acetylgalactosamine (GalNAc to the cell culture media. However, when key enzymes of the galactose/GalNAc salvage pathways were disrupted in tandem (GALE+GALK1 or GALE+GALK2, O-glycosylation was eliminated and could not be rescued by the addition of either galactose plus GalNAc or UDP-galactose plus UDP-GalNAc. GALK1 and GALK2 are key enzymes of the galactose/GalNAc salvage pathways. Mass spectrometry was performed on whole cell lysate of the knockout cell lines to verify the glycosylation phenotype. As expected, the GALE knockout was almost completely devoid of all O-glycosylation, with minimal glycosylation as a result of functional salvage pathways. However, the GALE+GALK1 and GALE+GALK2 knockout lines were devoid of all O-glycans. Mass spectrometry analysis revealed that the disruption of GALE, GALK1, and GALE+GALK2 had little effect on the N-glycome. But when GALE was knocked out in tandem with GALK1, N-glycans were exclusively of the high mannose type. Due to the well-characterized nature of these five knockout cell lines, they will likely prove useful for a wide variety of applications.

N-Linked Glycosylation is an Important Parameter for Optimal Selection of Cell Lines Producing Biopharmaceutical Human IgG

NARCIS (Netherlands)

van Berkel, Patrick H. C.; Gerritsen, Jolanda; Perdok, Gerrard; Valbjorn, Jesper; Vink, Tom; van de Winkel, Jan G. J.; Parren, Paul W. H. I.

2009-01-01

We studied the variations in N-linked glycosylation of human IgG molecules derived from 105 different stable cell lines each expressing one of the six different antibodies. Antibody expression was based on glutamine synthetase selection technology in suspension growing CHO-KISV cells. The glycans

Unraveling the Molecular Complexity of O-Glycosylated Endogenous (N-Terminal) pro-B-Type Natriuretic Peptide Forms in Blood Plasma of Patients with Severe Heart Failure.

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Halfinger, Bernhard; Hammerer-Lercher, Angelika; Amplatz, Benno; Sarg, Bettina; Kremser, Leopold; Lindner, Herbert H

2017-01-01

Currently, N-terminal pro-B-type natriuretic peptide (NT-proBNP) and its physiologically active counterpart, BNP, are most frequently used as biomarkers for diagnosis, prognosis, and disease monitoring of heart failure (HF). Commercial NT-proBNP and BNP immunoassays cross-react to varying degrees with unprocessed proBNP, which is also found in the circulation. ProBNP processing and immunoassay response are related to O-linked glycosylation of NT-proBNP and proBNP. There is a clear and urgent need to identify the glycosylation sites in the endogenously circulating peptides requested by the community to gain further insights into the different naturally occurring forms. The glycosylation sites of (NT-) proBNP (NT-proBNP and/or proBNP) were characterized in leftovers of heparinized plasma samples of severe HF patients (NT-proBNP: >10000 ng/L) by using tandem immunoaffinity purification, sequential exoglycosidase treatment for glycan trimming, β-elimination and Michael addition chemistry, as well as high-resolution nano-flow liquid chromatography electrospray multistage mass spectrometry. We describe 9 distinct glycosylation sites on circulating (NT-) proBNP in HF patients. Differentially glycosylated variants were detected based on highly accurate mass determination and multistage mass spectrometry. Remarkably, for each of the identified proteolytic glycopeptides, a nonglycosylated form also was detectable. Our results directly demonstrate for the first time a rather complex distribution of the endogenously circulating glycoforms by mass spectrometric analysis in HF patients, and show 9 glycosites in human (NT-) proBNP. This information may also have an impact on commercial immunoassays applying antibodies specific for the central region of (NT-) proBNP, which detect mostly nonglycosylated forms. © 2016 American Association for Clinical Chemistry.

Structure/functional aspects of the human riboflavin transporter-3 (SLC52A3): role of the predicted glycosylation and substrate-interacting sites.

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Subramanian, Veedamali S; Sabui, Subrata; Teafatiller, Trevor; Bohl, Jennifer A; Said, Hamid M

2017-08-01

The human riboflavin (RF) transporter-3 (hRFVT-3; product of the SLC52A3 gene) plays an essential role in the intestinal RF absorption process and is expressed exclusively at the apical membrane domain of polarized enterocytes. Previous studies have characterized different physiological/biological aspects of this transporter, but nothing is known about the glycosylation status of the hRFVT-3 protein and role of this modification in its physiology/biology. Additionally, little is known about the residues in the hRFVT-3 protein that interact with the ligand, RF. We addressed these issues using appropriate biochemical/molecular approaches, a protein-docking model, and established intestinal/renal epithelial cells. Our results showed that the hRFVT-3 protein is glycosylated and that glycosylation is important for its function. Mutating the predicted N -glycosylation sites at Asn 94 and Asn 168 led to a significant decrease in RF uptake; it also led to a marked intracellular (in the endoplasmic reticulum, ER) retention of the mutated proteins as shown by live-cell confocal imaging studies. The protein-docking model used in this study has identified a number of putative substrate-interacting sites: Ser 16 , Ile 20 , Trp 24 , Phe 142 , Thr 314 , and Asn 315 Mutating these potential interacting sites was indeed found to lead to a significant inhibition in RF uptake and to intracellular (ER) retention of the mutated proteins (except for the Phe 142 mutant). These results demonstrate that the hRFVT-3 protein is glycosylated and this glycosylation is important for its function and cell surface expression. This study also identified a number of residues in the hRFVT-3 polypeptide that are important for its function/cell surface expression.

Enzymatic glycosylation of multivalent scaffolds

Czech Academy of Sciences Publication Activity Database

Bojarová, Pavla; Rosencrantz, R. R.; Elling, L.; Křen, Vladimír

2013-01-01

Roč. 42, č. 11 (2013), s. 4774-4797 ISSN 0306-0012 R&D Projects: GA MŠk(CZ) LD13042; GA ČR GAP207/10/0321 Institutional support: RVO:61388971 Keywords : N-ACETYLGLUCOSAMINYLTRANSFERASE-III * MUCIN TANDEM REPEAT * NEIGHBORING RESIDUE GLYCOSYLATION Subject RIV: CC - Organic Chemistry Impact factor: 30.425, year: 2013

[The role of protein glycosylation in immune system].

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Ząbczyńska, Marta; Pocheć, Ewa

2015-01-01

Glycosylation is one of the most frequent post-translational modifications of proteins. The majority of cell surface and secreted proteins involved in immune response is glycosylated. The structural diversity of glycans depends on monosaccharide composition, type of glycosidic linkage and branching. These structural modifications determine a great variability of glycoproteins. The oligosaccharide components of proteins are regulated mostly by expression of glycosyltransferases and glycosidases and many environmental factors. Glycosylation influences the function of all immune cells. Glycans play a crucial role in intercellular contacts and leukocytes migration. These interactions are important in activation and proliferation of leukocytes and during immune response. The key immune proteins, such as TCR, MHC, TLR and antibodies are glycosylated. Sugars on the surface of pathogens and self-surface glycoproteins are recognized by special carbohydrate binding proteins called lectins. Changes of glycan structure are common in many pathological processes occurring in immune system, therefore they are used as molecular markers of different diseases.

Glycosylation of inositol phosphorylceramide sphingolipids is required for normal growth and reproduction in Arabidopsis

Energy Technology Data Exchange (ETDEWEB)

Tartaglio, Virginia [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Rennie, Emilie A. [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Univ. of Nebraska, Lincoln, NE (United States). Center for Plant Science Innovation and Dept. of Biochemistry; Cahoon, Rebecca [Univ. of Nebraska, Lincoln, NE (United States). Center for Plant Science Innovation and Dept. of Biochemistry; Wang, George [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Baidoo, Edward [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Mortimer, Jennifer C. [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Cahoon, Edgar B. [Univ. of Nebraska, Lincoln, NE (United States). Center for Plant Science Innovation and Dept. of Biochemistry; Scheller, Henrik V. [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Univ. of California, Berkeley, CA (United States). Dept. of Plant and Microbial Biology

2016-09-19

Sphingolipids are a major component of plant plasma membranes and endomembranes, and mediate a diverse range of biological processes. Study of the highly glycosylated glycosyl inositol phosphorylceramide (GIPC) sphingolipids has been slow as a result of challenges associated with the extractability of GIPCs, and their functions in the plant remain poorly characterized. We recently discovered an Arabidopsis GIPC glucuronosyltransferase, INOSITOL PHOSPHORYLCERAMIDE GLUCURONOSYLTRANSFERASE 1 (IPUT1), which is the first enzyme in the GIPC glycosylation pathway. Plants homozygous for the iput1 loss-of-function mutation were unobtainable, and so the developmental effects of reduced GIPC glucuronosylation could not be analyzed in planta. Using a pollen-specific rescue construct, we have here isolated homozygous iput1 mutants. The iput1 mutants show severe dwarfism, compromised pollen tube guidance, and constitutive activation of salicyclic acid-mediated defense pathways. The mutants also possess reduced GIPCs, increased ceramides, and an increased incorporation of short-chain fatty acids and dihydroxylated bases into inositol phosphorylceramides and GIPCs. The assignment of a direct role for GIPC glycan head groups in the impaired processes in iput1 mutants is complicated by the vast compensatory changes in the sphingolipidome; however, our results reveal that the glycosylation steps of GIPC biosynthesis are important regulated components of sphingolipid metabolism. In conclusion, this study corroborates previously suggested roles for GIPC glycans in plant growth and defense, suggests important role s for them in reproduction and demonstrates that the entire sphingolipidome is sensitive to their status.

Fab glycosylation of immunoglobulin G does not associate with improvement of rheumatoid arthritis during pregnancy.

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Bondt, Albert; Wuhrer, Manfred; Kuijper, T Martijn; Hazes, Johanna M W; Dolhain, Radboud J E M

2016-11-25

Changes in immunoglobulin G (IgG) constant domain (Fc) glycosylation are associated with changes in rheumatoid arthritis (RA) disease activity in response to pregnancy. Here, we sought to determine whether the same holds true for variable domain (Fab) glycosylation. IgGs were captured from RA and control sera obtained before (RA only), during and after pregnancy, followed by Fc and Fab separation, glycan release, and mass spectrometric detection. In parallel, glycans from intact IgG were analysed. The data was used to calculate glycosylation traits, and to estimate the level of Fab glycosylation. The overall level of Fab glycosylation was increased in RA patients compared to controls, while no differences in Fab glycosylation patterns were found. For the Fc and intact IgG (Total) previously observed differences in galactosylation and bisection were confirmed. Furthermore, increased galactosylation of Fc and Total were associated with lower disease activity and autoantibody positivity. In addition, the change in Fc galactosylation associated with the change in disease activity during pregnancy and after delivery, while this was not the case for Fab. In contrast to changes in Fc glycosylation, changes in Fab glycosylation are not associated with improvement of RA during pregnancy and arthritis flare after delivery.

Dengue Virus Glycosylation: What Do We Know?

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Sally S. L. Yap

2017-07-01

Full Text Available In many infectious diseases caused by either viruses or bacteria, pathogen glycoproteins play important roles during the infection cycle, ranging from entry to successful intracellular replication and host immune evasion. Dengue is no exception. Dengue virus glycoproteins, envelope protein (E and non-structural protein 1 (NS1 are two popular sub-unit vaccine candidates. E protein on the virion surface is the major target of neutralizing antibodies. NS1 which is secreted during DENV infection has been shown to induce a variety of host responses through its binding to several host factors. However, despite their critical role in disease and protection, the glycosylated variants of these two proteins and their biological importance have remained understudied. In this review, we seek to provide a comprehensive summary of the current knowledge on protein glycosylation in DENV, and its role in virus biogenesis, host cell receptor interaction and disease pathogenesis.

IN VITRO STUDY ON INHIBITION OF GLYCOSYLATION OF ...

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complications of diabetes mellitus (Makita et al., 1991). Apart from protein ... enzymes; inhibition of regulatory molecule binding; crosslinking of glycosylated .... further investigation specific bio active compound responsible for such activities.

Altered glycosylation of complexed native IgG molecules is associated with disease activity of systemic lupus erythematosus.

Science.gov (United States)

Sjöwall, C; Zapf, J; von Löhneysen, S; Magorivska, I; Biermann, M; Janko, C; Winkler, S; Bilyy, R; Schett, G; Herrmann, M; Muñoz, L E

2015-05-01

In addition to the redundancy of the receptors for the Fc portion of immunoglobulins, glycans result in potential ligands for a plethora of lectin receptors found in immune effector cells. Here we analysed the exposure of glycans containing fucosyl residues and the fucosylated tri-mannose N-type core by complexed native IgG in longitudinal serum samples of well-characterized patients with systemic lupus erythematosus. Consecutive serum samples of a cohort of 15 patients with systemic lupus erythematosus during periods of increased disease activity and remission were analysed. All patients fulfilled the 1982 American College of Rheumatology classification criteria. Sera of 15 sex- and age-matched normal healthy blood donors served as controls. The levels and type of glycosylation of complexed random IgG was measured with lectin enzyme-immunosorbent assays. After specifically gathering IgG complexes from sera, biotinylated lectins Aleuria aurantia lectin and Lens culinaris agglutinin were employed to detect IgG-associated fucosyl residues and the fucosylated tri-mannose N-glycan core, respectively. In sandwich-ELISAs, IgG-associated IgM, IgA, C1q, C3c and C-reactive protein (CRP) were detected as candidates for IgG immune complex constituents. We studied associations of the glycan of complexed IgG and disease activity according to the physician's global assessment of disease activity and the systemic lupus erythematosus disease activity index 2000 documented at the moment of blood taking. Our results showed significantly higher levels of Aleuria aurantia lectin and Lens culinaris agglutinin binding sites exposed on IgG complexes of patients with systemic lupus erythematosus than on those of normal healthy blood donors. Disease activity in systemic lupus erythematosus correlated with higher exposure of Aleuria aurantia lectin-reactive fucosyl residues by immobilized IgG complexes. Top levels of Aleuria aurantia lectin-reactivity were found in samples taken during the

Introduction of a glycosylation site in the constant region decreases the aggregation of adalimumab Fab.

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Nakamura, Hitomi; Oda-Ueda, Naoko; Ueda, Tadashi; Ohkuri, Takatoshi

2018-06-18

The production of therapeutic monoclonal antibodies is costly; therefore, antigen-binding fragments (Fabs) can be used instead. However, their tendency toward aggregation can reduce the half-life in the plasma and the therapeutic effectiveness. To examine the effect of glycosylation on the properties of the Fab of a therapeutic antibody, an N-glycosylation site was introduced at position 178 of the H-chain constant region of adalimumab Fab through site-directed mutagenesis of L178 N (H:L178 N Fab), and then H:L178 N Fab was expressed in Pichia pastoris. SDS-PAGE analysis with treatment of N-glycosidase F or periodic acid-Schiff reagent showed that H:L178 N Fab contained a relatively low glycan level. Moreover, the H:L178 N mutation did not decrease the binding activity and thermal stability of Fab, and H:L178 N Fab was more resistant to protease digestion than wild-type Fab. The aggregation of Fab induced by pH-shift stress was measured by monitoring the optical density at 350 nm. Although the wild-type Fab showed a large increase in optical density with an increase of protein concentration, no such increase of turbidity during aggregation was found in H:L178 N Fab. These results demonstrated that glycosylation at position 178 of the H-chain constant region of adalimumab Fab can prevent protein aggregation, and therefore serve as a potentially effective platform for drug development. Copyright © 2018. Published by Elsevier Inc.

Glucosilación no enzimática y complicaciones crónicas de la diabetes mellitus Non-enzymatic glycosylation and chronic complications of diabetes mellitus

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Jeddú Cruz Hernández

2010-08-01

Full Text Available INTRODUCCIÓN: la hiperglucemia es considerada hoy como un factor patogénico fundamental del desarrollo de las complicaciones neurovasculares diabéticas y, específicamente, desempeña un papel preponderante en el fenómeno de la glucosilación no enzimática y la formación de productos finales de la glucosilación avanzada. OBJETIVOS: describir los mecanismos de la formación de los productos finales de la glucosilación avanzada y su relación con las complicaciones de la diabetes mellitus. DESARROLLO: los productos finales de la glucosilación avanzada se producen por la reacción no enzimática de la glucosa y otros derivados glucados (glioxal, metilglioxal y 3-desoxiglucosona con grupos amino de proteínas de larga vida. La glucosilación altera la estructura, las propiedades físico-químicas y la función de las proteínas intracelulares y extracelulares. En la membrana basal de los pequeños vasos se produce un engrosamiento y una distorsión de su estructura, que ocasiona pérdida de la elasticidad de la pared vascular y una permeabilidad anormal de esta a las proteínas (disfunción endotelial, así como aumento de la génesis de especies reactivas del oxígeno. La unión de productos finales de la glucosilación avanzada a sus receptores de membrana favorece la producción citoquinas y factores de crecimiento por los macrófagos y células mesangiales. Todo lo anterior favorece al desarrollo de aterosclerosis. CONCLUSIONES: los productos finales de la glucosilación avanzada desempeñan un importante papel en el desarrollo de las complicaciones microvasculares y macrovasculares en el diabético. El control metabólico estricto de la glucemia y, en la actualidad, la terapéutica farmacológica con agentes que inhiben la formación de productos finales de la glucosilación avanzada o tienen acción antioxidante, constituyen alternativas terapéuticas para la prevención y solución del problema de las complicaciones crónicas de la

Aberrant Glycosylation in the Left Ventricle and Plasma of Rats with Cardiac Hypertrophy and Heart Failure.

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Chiaki Nagai-Okatani

Full Text Available Targeted proteomics focusing on post-translational modifications, including glycosylation, is a useful strategy for discovering novel biomarkers. To apply this strategy effectively to cardiac hypertrophy and resultant heart failure, we aimed to characterize glycosylation profiles in the left ventricle and plasma of rats with cardiac hypertrophy. Dahl salt-sensitive hypertensive rats, a model of hypertension-induced cardiac hypertrophy, were fed a high-salt (8% NaCl diet starting at 6 weeks. As a result, they exhibited cardiac hypertrophy at 12 weeks and partially impaired cardiac function at 16 weeks compared with control rats fed a low-salt (0.3% NaCl diet. Gene expression analysis revealed significant changes in the expression of genes encoding glycosyltransferases and glycosidases. Glycoproteome profiling using lectin microarrays indicated upregulation of mucin-type O-glycosylation, especially disialyl-T, and downregulation of core fucosylation on N-glycans, detected by specific interactions with Amaranthus caudatus and Aspergillus oryzae lectins, respectively. Upregulation of plasma α-l-fucosidase activity was identified as a biomarker candidate for cardiac hypertrophy, which is expected to support the existing marker, atrial natriuretic peptide and its related peptides. Proteomic analysis identified cysteine and glycine-rich protein 3, a master regulator of cardiac muscle function, as an O-glycosylated protein with altered glycosylation in the rats with cardiac hypertrophy, suggesting that alternations in O-glycosylation affect its oligomerization and function. In conclusion, our data provide evidence of significant changes in glycosylation pattern, specifically mucin-type O-glycosylation and core defucosylation, in the pathogenesis of cardiac hypertrophy and heart failure, suggesting that they are potential biomarkers for these diseases.

Control of mucin-type O-glycosylation

DEFF Research Database (Denmark)

Bennett, Eric P; Mandel, Ulla; Clausen, Henrik

2012-01-01

residues, is one of the most abundant forms of protein glycosylation in animals. Although most protein glycosylation is controlled by one or two genes encoding the enzymes responsible for the initiation of glycosylation, i.e. the step where the first glycan is attached to the relevant amino acid residue...... in the protein, mucin-type O-glycosylation is controlled by a large family of up to 20 homologous genes encoding UDP-GalNAc:polypeptide GalNAc-transferases (GalNAc-Ts) (EC 2.4.1.41). Therefore, mucin-type O-glycosylation has the greatest potential for differential regulation in cells and tissues. The Gal...

Kinetics of N-Glycan Release from Human Immunoglobulin G (IgG) by PNGase F: All Glycans Are Not Created Equal.

Science.gov (United States)

Huang, Yining; Orlando, Ron

2017-12-01

The biologic activity of IgG molecules is modulated by its crystallizable fragment N-glycosylation, and thus, the analysis of IgG glycosylation is critical. A standard approach to analyze glycosylation of IgGs involves the release of the N-glycans by the enzyme peptide N-glycosidase F, which cleaves the linkage between the asparagine residue and innermost N-acetylglucosamine (GlcNAc) of all N-glycans except those containing a 3-linked fucose attached to the reducing terminal GlcNAc residue. The importance of obtaining complete glycan release for accurate quantitation led us to investigate the kinetics of this de-glycosylation reaction for IgG glycopeptides and to determine the effect of glycan structure and amino acid sequence on the rate of glycan release from glycopeptides of IgGs. This study revealed that the slight differences in amino acid sequences did not lead to a statistically different deglycosylation rate. However, statistically significant differences in the deglycosylation rate constants were observed between glycopeptides differing only in glycan structure ( i.e. , nonfucosylated, fucosylated, bisecting-GlcNAc, sialylated, etc .). For example, a single sialic acid residue was found to decrease the rate by a factor of 3. Similar reductions in rate were associated with the presence of a bisecting-GlcNAc. We predict the differences in release kinetics can lead to significant quantitative variations of the glycosylation study of IgGs.

Diagnosis and activity assessment of immunoglobulin A nephropathy: current perspectives on noninvasive testing with aberrantly glycosylated immunoglobulin A-related biomarkers

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Suzuki Y

2014-10-01

Full Text Available Yusuke Suzuki,1 Hitoshi Suzuki,1 Yuko Makita,1 Akiko Takahata,1 Keiko Takahashi,1 Masahiro Muto,1 Yohei Sasaki,1 Atikemu Kelimu,1 Keiichi Matsuzaki,2 Hiroyuki Yanagawa,1 Keiko Okazaki,1 Yasuhiko Tomino1 1Division of Nephrology, Department of Internal Medicine, Juntendo University Faculty of Medicine, Tokyo, 2Kyoto University Health Service, Kyoto, Japan Abstract: Immunoglobulin (Ig A nephropathy (IgAN is the most common form of glomerular disease worldwide and is associated with a poor prognosis. Thus, development of a curative treatment and strategies for early diagnosis and treatment are urgently needed. Pathological analysis of renal biopsy is the gold standard for the diagnosis and assessment of disease activity; however, immediate and frequent assessment based on biopsy specimens is difficult. Therefore, a simple and safe alternative is desirable. On the other hand, it is now widely accepted that multi-hit steps, including production of aberrantly glycosylated serum IgA1 (first hit, and IgG or IgA autoantibodies that recognize glycan containing epitopes on glycosylated serum IgA1 (second hit and their subsequent immune complex formation (third hit and glomerular deposition (fourth hit, are required for continued progression of IgAN. Although the prognostic and predictive values of several markers have been discussed elsewhere, we recently developed a highly sensitive and specific diagnostic method by measuring serum levels of glycosylated serum IgA1 and related IgA immune complex. In addition, we confirmed a significant correlation between serum levels of these essential effector molecules and disease activity after treatment, suggesting that each can be considered as a practical surrogate marker of therapeutic effects in this slowly progressive disease. Such a noninvasive diagnostic and activity assessment method using these disease-oriented specific biomarkers may be useful in the early diagnosis of and intervention in IgAN, with

A zebrafish model of congenital disorders of glycosylation with phosphomannose isomerase deficiency reveals an early opportunity for corrective mannose supplementation

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Jaime Chu

2013-01-01

Individuals with congenital disorders of glycosylation (CDG have recessive mutations in genes required for protein N-glycosylation, resulting in multi-systemic disease. Despite the well-characterized biochemical consequences in these individuals, the underlying cellular defects that contribute to CDG are not well understood. Synthesis of the lipid-linked oligosaccharide (LLO, which serves as the sugar donor for the N-glycosylation of secretory proteins, requires conversion of fructose-6-phosphate to mannose-6-phosphate via the phosphomannose isomerase (MPI enzyme. Individuals who are deficient in MPI present with bleeding, diarrhea, edema, gastrointestinal bleeding and liver fibrosis. MPI-CDG patients can be treated with oral mannose supplements, which is converted to mannose-6-phosphate through a minor complementary metabolic pathway, restoring protein glycosylation and ameliorating most symptoms, although liver disease continues to progress. Because Mpi deletion in mice causes early embryonic lethality and thus is difficult to study, we used zebrafish to establish a model of MPI-CDG. We used a morpholino to block mpi mRNA translation and established a concentration that consistently yielded 13% residual Mpi enzyme activity at 4 days post-fertilization (dpf, which is within the range of MPI activity detected in fibroblasts from MPI-CDG patients. Fluorophore-assisted carbohydrate electrophoresis detected decreased LLO and N-glycans in mpi morphants. These deficiencies resulted in 50% embryonic lethality by 4 dpf. Multi-systemic abnormalities, including small eyes, dysmorphic jaws, pericardial edema, a small liver and curled tails, occurred in 82% of the surviving larvae. Importantly, these phenotypes could be rescued with mannose supplementation. Thus, parallel processes in fish and humans contribute to the phenotypes caused by Mpi depletion. Interestingly, mannose was only effective if provided prior to 24 hpf. These data provide insight into treatment efficacy

Antiangiogenic activity of 2-deoxy-D-glucose.

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Jaime R Merchan

2010-10-01

Full Text Available During tumor angiogenesis, endothelial cells (ECs are engaged in a number of energy consuming biological processes, such as proliferation, migration, and capillary formation. Since glucose uptake and metabolism are increased to meet this energy need, the effects of the glycolytic inhibitor 2-deoxy-D-glucose (2-DG on in vitro and in vivo angiogenesis were investigated.In cell culture, 2-DG inhibited EC growth, induced cytotoxicity, blocked migration, and inhibited actively forming but not established endothelial capillaries. Surprisingly, 2-DG was a better inhibitor of these EC properties than two more efficacious glycolytic inhibitors, 2-fluorodeoxy-D-glucose and oxamate. As an alternative to a glycolytic inhibitory mechanism, we considered 2-DG's ability to interfere with endothelial N-linked glycosylation. 2-DG's effects were reversed by mannose, an N-linked glycosylation precursor, and at relevant concentrations 2-DG also inhibited synthesis of the lipid linked oligosaccharide (LLO N-glycosylation donor in a mannose-reversible manner. Inhibition of LLO synthesis activated the unfolded protein response (UPR, which resulted in induction of GADD153/CHOP and EC apoptosis (TUNEL assay. Thus, 2-DG's effects on ECs appeared primarily due to inhibition of LLOs synthesis, not glycolysis. 2-DG was then evaluated in two mouse models, inhibiting angiogenesis in both the matrigel plug assay and the LH(BETAT(AG transgenic retinoblastoma model.In conclusion, 2-DG inhibits endothelial cell angiogenesis in vitro and in vivo, at concentrations below those affecting tumor cells directly, most likely by interfering with N-linked glycosylation rather than glycolysis. Our data underscore the importance of glucose metabolism on neovascularization, and demonstrate a novel approach for anti-angiogenic strategies.

Hallmarks of glycosylation in cancer.

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Munkley, Jennifer; Elliott, David J

2016-06-07

Aberrant glycosylation plays a fundamental role in key pathological steps of tumour development and progression. Glycans have roles in cancer cell signalling, tumour cell dissociation and invasion, cell-matrix interactions, angiogenesis, metastasis and immune modulation. Aberrant glycosylation is often cited as a 'hallmark of cancer' but is notably absent from both the original hallmarks of cancer and from the next generation of emerging hallmarks. This review discusses how glycosylation is clearly an enabling characteristic that is causally associated with the acquisition of all the hallmark capabilities. Rather than aberrant glycosylation being itself a hallmark of cancer, another perspective is that glycans play a role in every recognised cancer hallmark.

Clinical and biochemical presentation of siblings with COG-7 deficiency, a lethal multiple O- and N-glycosylation disorder.

NARCIS (Netherlands)

Spaapen, L.J.; Bakker, J.A.; Meer, S.B. van der; Sijstermans, H.J.; Steet, R.A.; Wevers, R.A.; Jaeken, J.

2005-01-01

Congenital disorders of glycosylation (CDG) represent a group of inherited multiorgan diseases caused by defects in the biosynthesis of glycoproteins. We report on two dysmorphic siblings with severe liver disease who died at the age of a few weeks. Increased activities of lysosomal enzymes in

Synthesis of hydrazide-functionalized hydrophilic polymer hybrid graphene oxide for highly efficient N-glycopeptide enrichment and identification by mass spectrometry.

Science.gov (United States)

Bai, Haihong; Pan, Yiting; Guo, Cong; Zhao, Xinyuan; Shen, Bingquan; Wang, Xinghe; Liu, Zeyuan; Cheng, Yuanguo; Qin, Weijie; Qian, Xiaohong

2017-08-15

Protein N-glycosylation is one of the most important post-translational modifications, participating in many key biological and pathological processes. Large-scale and precise identification of N-glycosylated proteins and peptides is especially beneficial for understanding their biological functions and for discovery of new clinical biomarkers and therapeutic drug targets. However, protein N-glycosylation is microheterogeneous and low abundant in living organisms, therefore specific enrichment of N-glycosylated proteins/peptides before mass spectrometry analysis is a prerequisite. In this work, we developed a new type of polymer hybrid graphene oxide (GO) by in situ growth of hydrazide-functionalized hydrophilic polymer chains on the GO surface (GO-PAAH) for selective N-glycopeptide enrichment and identification by mass spectrometry. The densely attached and low steric hindrance hydrazide groups as well as the highly hydrophilic nature of GO-PAAH facilitate N-glycopeptide enrichment by the combination of hydrazide capturing and HILIC interaction. Taking advantage of the unique features of GO-PAAH, all of the three N-glycopeptides of bovine fetuin were successfully enriched and identified with significantly enhanced signal intensities from a digest mixture of bovine fetuin and bovine serum albumin at a mass ratio of 1:100, demonstrating the excellent enrichment selectivity of GO-PAAH. Furthermore, a total of 507 N-glycosylation sites and 480 N-glycopeptides in 232 N-glycoproteins were enriched and identified from 10μL of human serum by three replicates using this novel enrichment material, which is nearly two times higher than the commercial hydrazide resin based method (280 N-glycosylation sites, 261 N-glycopeptides and 144 N-glycoproteins in three experiments). Among the identified, 95 N-glycosylation sites were not reported in the Uniprot database, and 106 N-glycoproteins were disease related in the Nextprot database, indicating the potential of this new

O-GLYCBASE: a revised database of O-glycosylated proteins

DEFF Research Database (Denmark)

Hansen, Jan; Lund, Ole; Nielsen, Jens O.

1996-01-01

O-GLYCBASE is a comprehensive database of information on glycoproteins and their O-linked glycosylation sites. Entries are compiled and revised from the SWISS-PROT and PIR databases as well as directly from recently published reports. Nineteen percent of the entries extracted from the databases n...... of mucin type O-glycosylation sites in mammalian glycoproteins exclusively from the primary sequence is made available by E-mail or WWW. The O-GLYCBASE database is also available electronically through our WWW server or by anonymous FTP....

Cell Surface Glycosylation Is Required for Efficient Mating of Haloferax volcanii

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Yarden Shalev

2017-07-01

Full Text Available Halophilic archaea use a fusion-based mating system for lateral gene transfer across cells, yet the molecular mechanisms involved remain unknown. Previous work implied that cell fusion involves cell–cell recognition since fusion occurs more efficiently between cells from the same species. Long believed to be restricted only to Eukarya, it is now known that cells of all three domains of life perform N-glycosylation, the covalent attachment of glycans to select target asparagine residues in proteins, and that this post-translational modification is common for archaeal cell surface proteins. Here, we show that differences in glycosylation of the Haloferax volcanii surface-layer glycoprotein, brought about either by changing medium salinity or by knocking out key glycosylation genes, reduced mating success. Thus, different glycosylation patterns are likely to underlie mating preference in halophilic archaea, contributing to speciation processes.

Neuraminidase stalk length and additional glycosylation of the hemagglutinin influence the virulence of influenza H5N1 viruses for mice.

Science.gov (United States)

Matsuoka, Yumiko; Swayne, David E; Thomas, Colleen; Rameix-Welti, Marie-Anne; Naffakh, Nadia; Warnes, Christine; Altholtz, Melanie; Donis, Ruben; Subbarao, Kanta

2009-05-01

Following circulation of avian influenza H5 and H7 viruses in poultry, the hemagglutinin (HA) can acquire additional glycosylation sites, and the neuraminidase (NA) stalk becomes shorter. We investigated whether these features play a role in the pathogenesis of infection in mammalian hosts. From 1996 to 2007, H5N1 viruses with a short NA stalk have become widespread in several avian species. Compared to viruses with a long-stalk NA, viruses with a short-stalk NA showed a decreased capacity to elute from red blood cells and an increased virulence in mice, but not in chickens. The presence of additional HA glycosylation sites had less of an effect on virulence than did NA stalk length. The short-stalk NA of H5N1 viruses circulating in Asia may contribute to virulence in humans.

Neuraminidase Stalk Length and Additional Glycosylation of the Hemagglutinin Influence the Virulence of Influenza H5N1 Viruses for Mice▿

Science.gov (United States)

Matsuoka, Yumiko; Swayne, David E.; Thomas, Colleen; Rameix-Welti, Marie-Anne; Naffakh, Nadia; Warnes, Christine; Altholtz, Melanie; Donis, Ruben; Subbarao, Kanta

2009-01-01

Following circulation of avian influenza H5 and H7 viruses in poultry, the hemagglutinin (HA) can acquire additional glycosylation sites, and the neuraminidase (NA) stalk becomes shorter. We investigated whether these features play a role in the pathogenesis of infection in mammalian hosts. From 1996 to 2007, H5N1 viruses with a short NA stalk have become widespread in several avian species. Compared to viruses with a long-stalk NA, viruses with a short-stalk NA showed a decreased capacity to elute from red blood cells and an increased virulence in mice, but not in chickens. The presence of additional HA glycosylation sites had less of an effect on virulence than did NA stalk length. The short-stalk NA of H5N1 viruses circulating in Asia may contribute to virulence in humans. PMID:19225004

THERAPEUTIC EFFECTS OF HIGHLY PURIFIED DE-GLYCOSYLATED GCMAF IN THE IMMUNOTHERAPY OF PATIENTS WITH CHRONIC DISEASES

OpenAIRE

Lynda Thyer; Emma Ward; Rodney Smith; Jacopo J.V. Branca; Gabriele Morucci; Massimo Gulisano; David Noakes; Stefania Pacini

2013-01-01

The de-Glycosylated vitamin D binding protein is a powerful Macrophage Activating Factor (GcMAF) that shows multiple biological effects that could be exploited in the immunotherapy of tumours, viral infections and autism. Here we report the observation of a series of clinical cases describing the results obtained administering highly purified GcMAF to patients with diverse types of chronic diseases. These are heterogeneous and refer to patients with different types of diseases at different s...

Role of Cytokine-Induced Glycosylation Changes in Regulating Cell Interactions and Cell Signaling in Inflammatory Diseases and Cancer

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Justine H. Dewald

2016-11-01

Full Text Available Glycosylation is one of the most important modifications of proteins and lipids, and cell surface glycoconjugates are thought to play important roles in a variety of biological functions including cell-cell and cell-substrate interactions, bacterial adhesion, cell immunogenicity and cell signaling. Alterations of glycosylation are observed in number of diseases such as cancer and chronic inflammation. In that context, pro-inflammatory cytokines have been shown to modulate cell surface glycosylation by regulating the expression of glycosyltransferases involved in the biosynthesis of carbohydrate chains. These changes in cell surface glycosylation are also known to regulate cell signaling and could contribute to disease pathogenesis. This review summarizes our current knowledge of the glycosylation changes induced by pro-inflammatory cytokines, with a particular focus on cancer and cystic fibrosis, and their consequences on cell interactions and signaling.

Glycosylation as a Main Regulator of Growth and Death Factor Receptors Signaling

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Inês Gomes Ferreira

2018-02-01

Full Text Available Glycosylation is a very frequent and functionally important post-translational protein modification that undergoes profound changes in cancer. Growth and death factor receptors and plasma membrane glycoproteins, which upon activation by extracellular ligands trigger a signal transduction cascade, are targets of several molecular anti-cancer drugs. In this review, we provide a thorough picture of the mechanisms bywhich glycosylation affects the activity of growth and death factor receptors in normal and pathological conditions. Glycosylation affects receptor activity through three non-mutually exclusive basic mechanisms: (1 by directly regulating intracellular transport, ligand binding, oligomerization and signaling of receptors; (2 through the binding of receptor carbohydrate structures to galectins, forming a lattice thatregulates receptor turnover on the plasma membrane; and (3 by receptor interaction with gangliosides inside membrane microdomains. Some carbohydrate chains, for example core fucose and β1,6-branching, exert a stimulatory effect on all receptors, while other structures exert opposite effects on different receptors or in different cellular contexts. In light of the crucial role played by glycosylation in the regulation of receptor activity, the development of next-generation drugs targeting glyco-epitopes of growth factor receptors should be considered a therapeutically interesting goal.

Glycogenomics as a mass spectrometry-guided genome-mining method for microbial glycosylated molecules.

Science.gov (United States)

Kersten, Roland D; Ziemert, Nadine; Gonzalez, David J; Duggan, Brendan M; Nizet, Victor; Dorrestein, Pieter C; Moore, Bradley S

2013-11-19

Glycosyl groups are an essential mediator of molecular interactions in cells and on cellular surfaces. There are very few methods that directly relate sugar-containing molecules to their biosynthetic machineries. Here, we introduce glycogenomics as an experiment-guided genome-mining approach for fast characterization of glycosylated natural products (GNPs) and their biosynthetic pathways from genome-sequenced microbes by targeting glycosyl groups in microbial metabolomes. Microbial GNPs consist of aglycone and glycosyl structure groups in which the sugar unit(s) are often critical for the GNP's bioactivity, e.g., by promoting binding to a target biomolecule. GNPs are a structurally diverse class of molecules with important pharmaceutical and agrochemical applications. Herein, O- and N-glycosyl groups are characterized in their sugar monomers by tandem mass spectrometry (MS) and matched to corresponding glycosylation genes in secondary metabolic pathways by a MS-glycogenetic code. The associated aglycone biosynthetic genes of the GNP genotype then classify the natural product to further guide structure elucidation. We highlight the glycogenomic strategy by the characterization of several bioactive glycosylated molecules and their gene clusters, including the anticancer agent cinerubin B from Streptomyces sp. SPB74 and an antibiotic, arenimycin B, from Salinispora arenicola CNB-527.

The glycosylated IgII extracellular domain of EMMPRIN is implicated in the induction of MMP-2.

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Papadimitropoulou, Adriana; Mamalaki, Avgi

2013-07-01

EMMPRIN is a widely expressed transmembrane glycoprotein that plays important roles in many physiological and pathological processes, such as tumor invasion and metastasis. It stimulates the production of matrix metalloproteinase (MMPs) by tumor-associated fibroblasts. In the present study, our aim was to (a) to investigate if the IgII loop domain of the extracellular domain (ECD) of EMMPRIN contributes to the MMP production by fibroblasts and (b) to evaluate the significance of glycosylation in this process. For this purpose, we expressed the ECD, IgI, or IgII domains of EMMPRIN, in their glycosylated and non-glycosylated forms, in the heterologous expression systems of P. pastoris and E. coli, respectively. Dermal fibroblasts were treated with purified recombinant domains and proteins from cell extracts and supernatants were analyzed by Western blot and zymography assays. Fibroblasts treated with ECD-, IgI-, and IgII-glycosylated domains of EMMPRIN significantly stimulated the gelatinolytic activity of MMP-2, compared to untreated fibroblasts, whereas no significant effect was observed after treatment with the non-glycosylated ECD, IgI, and IgII domains. Western blot analysis from cell extracts and supernatants revealed that only the glycosylated forms were able to stimulate MMP-2 production and secretion, respectively. Quantitative PCR revealed that this effect was not attributed to transcriptional alterations. This study showed that N-glycosylation was a prerequisite for efficient MMP-2 production, with the IgII loop domain contributing significantly to this process. Perturbation of the function of IgII-EMMPRIN loop could have potential therapeutic value in the inhibition of MMP-2-dependent cancer cell invasion and metastasis.

The effect of glycosylation on cytotoxicity of Ibaraki virus nonstructural protein NS3

Science.gov (United States)

URATA, Maho; WATANABE, Rie; IWATA, Hiroyuki

2015-01-01

The cytotoxicity of Ibaraki virus nonstructural protein NS3 was confirmed, and the contribution of glycosylation to this activity was examined by using glycosylation mutants of NS3 generated by site-directed mutagenesis. The expression of NS3 resulted in leakage of lactate dehydrogenase to the culture supernatant, suggesting the cytotoxicity of this protein. The lack of glycosylation impaired the transport of NS3 to the plasma membrane and resulted in reduced cytotoxicity. Combined with the previous observation that NS3 glycosylation was specifically observed in mammalian cells (Urata et al., Virus Research 2014), it was suggested that the alteration of NS3 cytotoxicity through modulating glycosylation is one of the strategies to achieve host specific pathogenisity of Ibaraki virus between mammals and vector arthropods. PMID:26178820

Cell culture media supplementation of infrequently used sugars for the targeted shifting of protein glycosylation profiles.

Science.gov (United States)

Hossler, Patrick; Racicot, Christopher; Chumsae, Christopher; McDermott, Sean; Cochran, Keith

2017-03-01

Mammalian cells in culture rely on sources of carbohydrates to supply the energy requirements for proliferation. In addition, carbohydrates provide a large source of the carbon supply for supporting various other metabolic activities, including the intermediates involved in the protein glycosylation pathway. Glucose and galactose, in particular, are commonly used sugars in culture media for these purposes. However, there exists a very large repertoire of other sugars in nature, and many that have been chemically synthesized. These sugars are particularly interesting because they can be utilized by cells in culture in distinct ways. In the present work it has been found that many infrequently used sugars, and the corresponding cellular response towards them as substrates, led to differences in the protein N-glycosylation profile of a recombinant glycoprotein. The selective media supplementation of raffinose, trehalose, turanose, palatinose, melezitose, psicose, lactose, lactulose, and mannose were found to be capable of redirecting N-glycan oligosaccharide profiles. Despite this shifting of protein glycosylation, there were no other adverse changes in culture performance, including both cell growth and cellular productivity over a wide range of supplemented sugar concentrations. The approach presented highlights a potential means towards both the targeted shifting of protein glycosylation profiles and ensuring recombinant protein comparability, which up to this point in time has remained under-appreciated for these under-utilized compounds. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:511-522, 2017. © 2017 American Institute of Chemical Engineers.

Characterization of the N-linked glycosylation site of recombinant pectate lyase

NARCIS (Netherlands)

Colangelo, J.; Licon, V.; Benen, J.A.E.; Visser, J.; Bergmann, C.; Orlando, R.

1999-01-01

Recombinant pectate lyase from Aspergillus niger was overexpressed in Aspergillus nidulans. The two recombinant proteins produced differed in molecular mass by 1200 Da, which suggested that the larger molecular weight protein was glycosylated. The deduced amino acid sequence was searched for

Does Physical Activity Mediate the Associations Between Local-Area Descriptive Norms, Built Environment Walkability, and Glycosylated Hemoglobin?

Science.gov (United States)

Carroll, Suzanne J; Niyonsenga, Theo; Coffee, Neil T; Taylor, Anne W; Daniel, Mark

2017-08-23

Associations between local-area residential features and glycosylated hemoglobin (HbA 1c ) may be mediated by individual-level health behaviors. Such indirect effects have rarely been tested. This study assessed whether individual-level self-reported physical activity mediated the influence of local-area descriptive norms and objectively expressed walkability on 10-year change in HbA 1c . HbA 1c was assessed three times for adults in a 10-year population-based biomedical cohort ( n = 4056). Local-area norms specific to each participant were calculated, aggregating responses from a separate statewide surveillance survey for 1600 m road-network buffers centered on participant addresses (local prevalence of overweight/obesity (body mass index ≥25 kg/m²) and physical inactivity (Walkability was directly and indirectly protective of worsening HbA 1c . Local-area descriptive norms and walkability influence cardiometabolic risk trajectory through individual-level physical activity. Efforts to reduce population cardiometabolic risk should consider the extent of local-area unhealthful behavioral norms and walkability in tailoring strategies to improve physical activity.

Glycosylation of Candida albicans cell wall proteins is critical for induction of innate immune responses and apoptosis of epithelial cells.

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Jeanette Wagener

Full Text Available C. albicans is one of the most common fungal pathogen of humans, causing local and superficial mucosal infections in immunocompromised individuals. Given that the key structure mediating host-C. albicans interactions is the fungal cell wall, we aimed to identify features of the cell wall inducing epithelial responses and be associated with fungal pathogenesis. We demonstrate here the importance of cell wall protein glycosylation in epithelial immune activation with a predominant role for the highly branched N-glycosylation residues. Moreover, these glycan moieties induce growth arrest and apoptosis of epithelial cells. Using an in vitro model of oral candidosis we demonstrate, that apoptosis induction by C. albicans wild-type occurs in early stage of infection and strongly depends on intact cell wall protein glycosylation. These novel findings demonstrate that glycosylation of the C. albicans cell wall proteins appears essential for modulation of epithelial immunity and apoptosis induction, both of which may promote fungal pathogenesis in vivo.

A compound heterozygous mutation in DPAGT1 results in a congenital disorder of glycosylation with a relatively mild phenotype

NARCIS (Netherlands)

Iqbal, Z.; Shahzad, M.; Vissers, L.E.L.M.; Scherpenzeel, M. van; Gilissen, C.; Razzaq, A.; Zahoor, M.Y.; Khan, S.N.; Kleefstra, T.; Veltman, J.A.; Brouwer, A.P.M. de; Lefeber, D.J.; Bokhoven, H. van; Riazuddin, S.

2013-01-01

Congenital disorders of glycosylation (CDG) are a large group of recessive multisystem disorders caused by impaired protein or lipid glycosylation. The CDG-I subgroup is characterized by protein N-glycosylation defects originating in the endoplasmic reticulum. The genetic defect is known for 17

Diagnostic serum glycosylation profile in patients with intellectual disability as a result of MAN1B1 deficiency

DEFF Research Database (Denmark)

Van Scherpenzeel, Monique; Timal, Sharita; Rymen, Daisy

2014-01-01

Congenital disorders of glycosylation comprise a group of genetic defects with a high frequency of intellectual disability, caused by deficient glycosylation of proteins and lipids. The molecular basis of the majority of the congenital disorders of glycosylation type I subtypes, localized...... in the cytosol and endoplasmic reticulum, has been solved. However, elucidation of causative genes for defective Golgi glycosylation (congenital disorders of glycosylation type II) remains challenging because of a lack of sufficiently specific diagnostic serum methods. In a single patient with intellectual...... disability, whole-exome sequencing revealed MAN1B1 as congenital disorder of glycosylation type II candidate gene. A novel mass spectrometry method was applied for high-resolution glycoprofiling of intact plasma transferrin. A highly characteristic glycosylation signature was observed with hybrid type N...

Structural Analysis of β-Fructofuranosidase from Xanthophyllomyces dendrorhous Reveals Unique Features and the Crucial Role of N-Glycosylation in Oligomerization and Activity*

Science.gov (United States)

Ramírez-Escudero, Mercedes; Gimeno-Pérez, María; González, Beatriz; Linde, Dolores; Merdzo, Zoran; Fernández-Lobato, María; Sanz-Aparicio, Julia

2016-01-01

Xanthophyllomyces dendrorhous β-fructofuranosidase (XdINV)is a highly glycosylated dimeric enzyme that hydrolyzes sucrose and releases fructose from various fructooligosaccharides (FOS) and fructans. It also catalyzes the synthesis of FOS, prebiotics that stimulate the growth of beneficial bacteria in human gut. In contrast to most fructosylating enzymes, XdINV produces neo-FOS, which makes it an interesting biotechnology target. We present here its three-dimensional structure, which shows the expected bimodular arrangement and also a long extension of its C terminus that together with an N-linked glycan mediate the formation of an unusual dimer. The two active sites of the dimer are connected by a long crevice, which might indicate its potential ability to accommodate branched fructans. This arrangement could be representative of a group of GH32 yeast enzymes having the traits observed in XdINV. The inactive D80A mutant was used to obtain complexes with relevant substrates and products, with their crystals structures showing at least four binding subsites at each active site. Moreover, two different positions are observed from subsite +2 depending on the substrate, and thus, a flexible loop (Glu-334–His-343) is essential in binding sucrose and β(2–1)-linked oligosaccharides. Conversely, β(2–6) and neo-type substrates are accommodated mainly by stacking to Trp-105, explaining the production of neokestose and the efficient fructosylating activity of XdINV on α-glucosides. The role of relevant residues has been investigated by mutagenesis and kinetics measurements, and a model for the transfructosylating reaction has been proposed. The plasticity of its active site makes XdINV a valuable and flexible biocatalyst to produce novel bioconjugates. PMID:26823463

Yeast carboxypeptidase Y requires glycosylation for efficient intracellular transport, but not for vacuolar sorting, in vivo stability, or activity

DEFF Research Database (Denmark)

Winther, Jakob R.; Stevens, T H; Kielland-Brandt, Morten

1991-01-01

the formation of asparagine-linked glycosylation, had a similar effect on the transport of CPY at 23 degrees C. However, the absence of N-linked carbohydrate in general had the more dramatic result of blocking the transport of CPY altogether at an increased temperature (37 degrees C). The unglycosylated mutant...

A Crude 1-DNJ Extract from Home Made Bombyx Batryticatus Inhibits Diabetic Cardiomyopathy-Associated Fibrosis in db/db Mice and Reduces Protein N-Glycosylation Levels

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Qing Zhao

2018-06-01

Full Text Available The traditional Chinese drug Bombyx Batryticatus (BB, which is also named the white stiff silkworm, has been widely used in Chinese clinics for thousands of years. It is famous for its antispasmodic and blood circulation-promoting effects. Cardiomyocyte hypertrophy, interstitial cell hyperplasia, and myocardial fibrosis are closely related to the N-glycosylation of key proteins. To examine the alterations of N-glycosylation that occur in diabetic myocardium during the early stage of the disease, and to clarify the therapeutic effect of 1-Deoxynojirimycin (1-DNJ extracted from BB, we used the db/db (diabetic mouse model and an approach based on hydrophilic chromatography solid-phase extraction integrated with an liquid Chromatograph Mass Spectrometer (LC-MS identification strategy to perform a site-specific N-glycosylation analysis of left ventricular cardiomyocyte proteins. Advanced glycation end products (AGEs, hydroxyproline, connective tissue growth factor (CTGF, and other serum biochemical indicators were measured with enzyme-linked immunosorbent assays (ELISA. In addition, the α-1,6-fucosylation of N-glycans was profiled with lens culinaris agglutinin (LCA lectin blots and fluorescein isothiocyanate (FITC-labelled lectin affinity histochemistry. The results indicated that 1-DNJ administration obviously downregulated myocardium protein N-glycosylation in db/db mice. The expression levels of serum indicators and fibrosis-related cytokines were reduced significantly by 1-DNJ in a dose-dependent manner. The glycan α-1,6-fucosylation level of the db/db mouse myocardium was elevated, and the intervention effect of 1-DNJ administration on N-glycan α-1,6-fucosylation was significant. To verify this result, the well-known transforming growth factor-β (TGF-β/Smad2/3 pathway was selected, and core α-1,6-fucosylated TGF-β receptor II (TGFR-βII was analysed semi-quantitatively with western blotting. The result supported the conclusions obtained

Crystallization and diffraction analysis of β-N-acetylhexosaminidase from Aspergillus oryzae

International Nuclear Information System (INIS)

Vaněk, Ondřej; Brynda, Jiří; Hofbauerová, Kateřina; Kukačka, Zdeněk; Pachl, Petr; Bezouška, Karel; Řezáčová, Pavlína

2011-01-01

The fungal β-N-acetylhexosaminidase from A. oryzae was crystallized and diffraction data were collected from two crystal forms to 3.2 and 2.4 Å resolution, respectively. Fungal β-N-acetylhexosaminidases are enzymes that are used in the chemoenzymatic synthesis of biologically interesting oligosaccharides. The enzyme from Aspergillus oryzae was produced and purified from its natural source and crystallized using the hanging-drop vapour-diffusion method. Diffraction data from two crystal forms (primitive monoclinic and primitive tetragonal) were collected to resolutions of 3.2 and 2.4 Å, respectively. Electrophoretic and quantitative N-terminal protein-sequencing analyses confirmed that the crystals are formed by a complete biologically active enzyme consisting of a glycosylated catalytic unit and a noncovalently attached propeptide

Amino acid and glucose metabolism in fed-batch CHO cell culture affects antibody production and glycosylation.

Science.gov (United States)

Fan, Yuzhou; Jimenez Del Val, Ioscani; Müller, Christian; Wagtberg Sen, Jette; Rasmussen, Søren Kofoed; Kontoravdi, Cleo; Weilguny, Dietmar; Andersen, Mikael Rørdam

2015-03-01

Fed-batch Chinese hamster ovary (CHO) cell culture is the most commonly used process for IgG production in the biopharmaceutical industry. Amino acid and glucose consumption, cell growth, metabolism, antibody titer, and N-glycosylation patterns are always the major concerns during upstream process optimization, especially media optimization. Gaining knowledge on their interrelations could provide insight for obtaining higher immunoglobulin G (IgG) titer and better controlling glycosylation-related product quality. In this work, different fed-batch processes with two chemically defined proprietary media and feeds were studied using two IgG-producing cell lines. Our results indicate that the balance of glucose and amino acid concentration in the culture is important for cell growth, IgG titer and N-glycosylation. Accordingly, the ideal fate of glucose and amino acids in the culture could be mainly towards energy and recombinant product, respectively. Accumulation of by-products such as NH4(+) and lactate as a consequence of unbalanced nutrient supply to cell activities inhibits cell growth. The levels of Leu and Arg in the culture, which relate to cell growth and IgG productivity, need to be well controlled. Amino acids with the highest consumption rates correlate with the most abundant amino acids present in the produced IgG, and thus require sufficient availability during culture. Case-by-case analysis is necessary for understanding the effect of media and process optimization on glycosylation. We found that in certain cases the presence of Man5 glycan can be linked to limitation of UDP-GlcNAc biosynthesis as a result of insufficient extracellular Gln. However, under different culture conditions, high Man5 levels can also result from low α-1,3-mannosyl-glycoprotein 2-β-N-acetylglucosaminyltransferase (GnTI) and UDP-GlcNAc transporter activities, which may be attributed to high level of NH4+ in the cell culture. Furthermore, galactosylation of the mAb Fc glycans

Chiral reagents in glycosylation and modification of carbohydrates.

Science.gov (United States)

Wang, Hao-Yuan; Blaszczyk, Stephanie A; Xiao, Guozhi; Tang, Weiping

2018-02-05

Carbohydrates play a significant role in numerous biological events, and the chemical synthesis of carbohydrates is vital for further studies to understand their various biological functions. Due to the structural complexity of carbohydrates, the stereoselective formation of glycosidic linkages and the site-selective modification of hydroxyl groups are very challenging and at the same time extremely important. In recent years, the rapid development of chiral reagents including both chiral auxiliaries and chiral catalysts has significantly improved the stereoselectivity for glycosylation reactions and the site-selectivity for the modification of carbohydrates. These new tools will greatly facilitate the efficient synthesis of oligosaccharides, polysaccharides, and glycoconjugates. In this tutorial review, we will summarize these advances and highlight the most recent examples.

Stabilization of bacterially expressed erythropoietin by single site-specific introduction of short branched PEG chains at naturally occurring glycosylation sites.

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Hoffmann, E; Streichert, K; Nischan, N; Seitz, C; Brunner, T; Schwagerus, S; Hackenberger, C P R; Rubini, M

2016-05-24

The covalent attachment of polyethylene glycol (PEG) to therapeutic proteins can improve their physicochemical properties. In this work we utilized the non-natural amino acid p-azidophenylalanine (pAzF) in combination with the chemoselective Staudinger-phosphite reaction to install branched PEG chains to recombinant unglycosylated erythropoietin (EPO) at each single naturally occurring glycosylation site. PEGylation with two short 750 or 2000 Da PEG units at positions 24, 38, or 83 significantly decreased unspecific aggregation and proteolytic degradation while biological activity in vitro was preserved or even increased in comparison to full-glycosylated EPO. This site-specific bioconjugation approach permits to analyse the impact of PEGylation at single positions. These results represent an important step towards the engineering of site-specifically modified EPO variants from bacterial expression with increased therapeutic efficacy.

Prediction of mucin-type O-glycosylation sites in mammalian proteins using the composition of k-spaced amino acid pairs

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Sheng Zhi-Ya

2008-02-01

Full Text Available Abstract Background As one of the most common protein post-translational modifications, glycosylation is involved in a variety of important biological processes. Computational identification of glycosylation sites in protein sequences becomes increasingly important in the post-genomic era. A new encoding scheme was employed to improve the prediction of mucin-type O-glycosylation sites in mammalian proteins. Results A new protein bioinformatics tool, CKSAAP_OGlySite, was developed to predict mucin-type O-glycosylation serine/threonine (S/T sites in mammalian proteins. Using the composition of k-spaced amino acid pairs (CKSAAP based encoding scheme, the proposed method was trained and tested in a new and stringent O-glycosylation dataset with the assistance of Support Vector Machine (SVM. When the ratio of O-glycosylation to non-glycosylation sites in training datasets was set as 1:1, 10-fold cross-validation tests showed that the proposed method yielded a high accuracy of 83.1% and 81.4% in predicting O-glycosylated S and T sites, respectively. Based on the same datasets, CKSAAP_OGlySite resulted in a higher accuracy than the conventional binary encoding based method (about +5.0%. When trained and tested in 1:5 datasets, the CKSAAP encoding showed a more significant improvement than the binary encoding. We also merged the training datasets of S and T sites and integrated the prediction of S and T sites into one single predictor (i.e. S+T predictor. Either in 1:1 or 1:5 datasets, the performance of this S+T predictor was always slightly better than those predictors where S and T sites were independently predicted, suggesting that the molecular recognition of O-glycosylated S/T sites seems to be similar and the increase of the S+T predictor's accuracy may be a result of expanded training datasets. Moreover, CKSAAP_OGlySite was also shown to have better performance when benchmarked against two existing predictors. Conclusion Because of CKSAAP

Altered trafficking and unfolded protein response induction as a result of M3 muscarinic receptor impaired N-glycosylation.

Science.gov (United States)

Romero-Fernandez, Wilber; Borroto-Escuela, Dasiel O; Alea, Mileidys Perez; Garcia-Mesa, Yoelvis; Garriga, Pere

2011-12-01

The human M(3) muscarinic acetylcholine receptor is present in both the central and peripheral nervous system, and it is involved in the pathophysiology of several neurodegenerative and autoimmune diseases. We suggested a possible N-glycosylation map for the M(3) muscarinic receptor expressed in COS-7 cells. Here, we examined the role that N-linked glycans play in the folding and in the cell surface trafficking of this receptor. The five potential asparagine-linked glycosylation sites in the muscarinic receptor were mutated and transiently expressed in COS-7 cells. The elimination of N-glycan attachment sites did not affect the cellular expression levels of the receptor. However, proper receptor localization to the plasma membrane was affected as suggested by reduced [(3)H]-N-methylscopolamine binding. Confocal microscopy confirmed this observation and showed that the nonglycosylated receptor was primarily localized in the intracellular compartments. The mutant variant showed an increase in phosphorylation of the α-subunit of eukaryote initiation factor 2, and other well-known endoplasmic reticulum stress markers of the unfolded protein response pathway, which further supports the proposal of the improper intracellular accumulation of the nonglycosylated receptor. The receptor devoid of glycans showed more susceptibility to events that culminate in apoptosis reducing cell viability. Our findings suggest up-regulation of pro-apoptotic Bax protein, down-regulation of anti-apoptotic Bcl-2, and cleavage of caspase-3 effectors. Collectively, our data provide experimental evidence of the critical role that N-glycan chains play in determining muscarinic receptor distribution, localization, as well as cell integrity. © The Author 2011. Published by Oxford University Press. All rights reserved.

Glucosamine derived DISAL donors for stereoselective glycosylations under neutral conditions

DEFF Research Database (Denmark)

Grathe, S.; Thygesen, M.B.; Larsen, K.

2005-01-01

DISAL (methyl 3,5-dinitrosa/icylate) D-glcosyl, D-galactosyl, D-mannosyl, and L-quinovosyl donors have previously provided the efficient glycosylation of a range of substrates under either strictly neutral, mildly basic, or very mildly Lewis acidic (LiClO4) conditions. Herein we report the synthe......DISAL (methyl 3,5-dinitrosa/icylate) D-glcosyl, D-galactosyl, D-mannosyl, and L-quinovosyl donors have previously provided the efficient glycosylation of a range of substrates under either strictly neutral, mildly basic, or very mildly Lewis acidic (LiClO4) conditions. Herein we report...... the synthesis of new glucosamine DISAL donors, carrying N-TCP, -Troc, or -TFAc protecting groups, and their use in beta-(1,2-trans) selective glycosylations, primarily in NMP in the absence of any added Lewis acids, or in CH3NO2 with LiClO4. Finally, precise microwave heating proved effective in promoting...

Engineering Mammalian Mucin-type O-Glycosylation in Plants

DEFF Research Database (Denmark)

Yang, Zhang; Drew, Damian P; Jørgensen, Bodil

2012-01-01

-glycans are attached to proteins, and which structures are formed, difficult. Because plants are devoid of GalNAc-type O-glycosylation, we have assessed requirements for establishing human GalNAc O-glycosylation de novo in plants with the aim of developing cell systems with custom-designed O-glycosylation capacity...... was glycosylated with up to three and five GalNAc residues when co-expressed with GalNAc-T2 and a combination of GalNAc-T2 and GalNAc-T4, respectively, as determined by mass spectrometry. O-Glycosylation was furthermore demonstrated on a tandem repeat of MUC16 and interferon a2b. In plants, prolines in certain...... classes of proteins are hydroxylated and further substituted with plant-specific O-glycosylation; unsubstituted hydroxyprolines were identified in our MUC1 construct. In summary, this study demonstrates that mammalian type O-glycosylation can be established in plants and that plants may serve as a host...

Re-emergence of H3N2 strains carrying potential neutralizing mutations at the N-linked glycosylation site at the hemagglutinin head, post the 2009 H1N1 pandemic.

Science.gov (United States)

Ushirogawa, Hiroshi; Naito, Tadasuke; Tokunaga, Hirotoshi; Tanaka, Toshihiro; Nakano, Takashi; Terada, Kihei; Ohuchi, Masanobu; Saito, Mineki

2016-08-08

Seasonally prevalent H1N1 and H3N2 influenza A viruses have evolved by antigenic drift; this evolution has resulted in the acquisition of asparagine (N)-linked glycosylation sites (NGSs) in the globular head of hemagglutinin (HA), thereby affecting the antigenic and receptor-binding properties, as well as virulence. An epidemiological survey indicated that although the traditional seasonal H1N1 strain had disappeared, H3N2 became predominant again in the seasons (2010-11 and 2011-12) immediately following the H1N1 pandemic of 2009. Interestingly, although the 2009 pandemic H1N1 strain (H1N1pdm09) lacks additional NGSs, clinically isolated H3N2 strains obtained during these seasons gained N (Asn) residues at positions 45 and 144 of HA that forms additional NGSs. To investigate whether these NGSs are associated with re-emergence of H3N2 within the subtype, we tested the effect of amino acid substitutions on neutralizing activity by using the antisera raised against H3N2 strains with or without additional NGSs. Furthermore, because the N residue at position 144 of HA was identified as the site of mismatch between the vaccine and epidemic strains of 2011-2012, we generated mutant viruses by reverse genetics and tested the functional importance of this particular NGS for antibody-mediated neutralization by intranasal inoculation of mice. The results indicated that amino acid substitution at residue 144 significantly affected neutralization activity, acting as an escape mutation. Our data suggest that the newly acquired NGSs in the HA globular head may play an important role in the re-emergence of endemic seasonal H3N2 strain by aiding the escape from humoral immunity.

Macrocyclic bis-thioureas catalyze stereospecific glycosylation reactions.

Science.gov (United States)

Park, Yongho; Harper, Kaid C; Kuhl, Nadine; Kwan, Eugene E; Liu, Richard Y; Jacobsen, Eric N

2017-01-13

Carbohydrates are involved in nearly all aspects of biochemistry, but their complex chemical structures present long-standing practical challenges to their synthesis. In particular, stereochemical outcomes in glycosylation reactions are highly dependent on the steric and electronic properties of coupling partners; thus, carbohydrate synthesis is not easily predictable. Here we report the discovery of a macrocyclic bis-thiourea derivative that catalyzes stereospecific invertive substitution pathways of glycosyl chlorides. The utility of the catalyst is demonstrated in the synthesis of trans-1,2-, cis-1,2-, and 2-deoxy-β-glycosides. Mechanistic studies are consistent with a cooperative mechanism in which an electrophile and a nucleophile are simultaneously activated to effect a stereospecific substitution reaction. Copyright © 2017, American Association for the Advancement of Science.

A computational framework for the automated construction of glycosylation reaction networks.

Science.gov (United States)

Liu, Gang; Neelamegham, Sriram

2014-01-01

Glycosylation is among the most common and complex post-translational modifications identified to date. It proceeds through the catalytic action of multiple enzyme families that include the glycosyltransferases that add monosaccharides to growing glycans, and glycosidases which remove sugar residues to trim glycans. The expression level and specificity of these enzymes, in part, regulate the glycan distribution or glycome of specific cell/tissue systems. Currently, there is no systematic method to describe the enzymes and cellular reaction networks that catalyze glycosylation. To address this limitation, we present a streamlined machine-readable definition for the glycosylating enzymes and additional methodologies to construct and analyze glycosylation reaction networks. In this computational framework, the enzyme class is systematically designed to store detailed specificity data such as enzymatic functional group, linkage and substrate specificity. The new classes and their associated functions enable both single-reaction inference and automated full network reconstruction, when given a list of reactants and/or products along with the enzymes present in the system. In addition, graph theory is used to support functions that map the connectivity between two or more species in a network, and that generate subset models to identify rate-limiting steps regulating glycan biosynthesis. Finally, this framework allows the synthesis of biochemical reaction networks using mass spectrometry (MS) data. The features described above are illustrated using three case studies that examine: i) O-linked glycan biosynthesis during the construction of functional selectin-ligands; ii) automated N-linked glycosylation pathway construction; and iii) the handling and analysis of glycomics based MS data. Overall, the new computational framework enables automated glycosylation network model construction and analysis by integrating knowledge of glycan structure and enzyme biochemistry. All

A computational framework for the automated construction of glycosylation reaction networks.

Directory of Open Access Journals (Sweden)

Gang Liu

Full Text Available Glycosylation is among the most common and complex post-translational modifications identified to date. It proceeds through the catalytic action of multiple enzyme families that include the glycosyltransferases that add monosaccharides to growing glycans, and glycosidases which remove sugar residues to trim glycans. The expression level and specificity of these enzymes, in part, regulate the glycan distribution or glycome of specific cell/tissue systems. Currently, there is no systematic method to describe the enzymes and cellular reaction networks that catalyze glycosylation. To address this limitation, we present a streamlined machine-readable definition for the glycosylating enzymes and additional methodologies to construct and analyze glycosylation reaction networks. In this computational framework, the enzyme class is systematically designed to store detailed specificity data such as enzymatic functional group, linkage and substrate specificity. The new classes and their associated functions enable both single-reaction inference and automated full network reconstruction, when given a list of reactants and/or products along with the enzymes present in the system. In addition, graph theory is used to support functions that map the connectivity between two or more species in a network, and that generate subset models to identify rate-limiting steps regulating glycan biosynthesis. Finally, this framework allows the synthesis of biochemical reaction networks using mass spectrometry (MS data. The features described above are illustrated using three case studies that examine: i O-linked glycan biosynthesis during the construction of functional selectin-ligands; ii automated N-linked glycosylation pathway construction; and iii the handling and analysis of glycomics based MS data. Overall, the new computational framework enables automated glycosylation network model construction and analysis by integrating knowledge of glycan structure and enzyme

Chemical constituents and biological activities of Dianthus elegans var. elegans.

Science.gov (United States)

Mutlu, Kiymet; Sarikahya, Nazli Boke; Nalbantsoy, Ayse; Kirmizigul, Suheyla

2018-06-01

Chemical investigation of the aerial parts of Dianthus elegans var. elegans afforded two previously undescribed saponins, named dianosides M-N (1-2), together with four oleanane-type triterpenoid glycosides (3-6). Their structures were elucidated as 3-O-α-L-arabinofuranosyl-16α-hydroxyolean-12-ene-23α, 28β-dioic acid (1) and 3-O-α-L-arabinofuranosyl-(1 → 3)-β-D-glucopyranosyl 16α-hydroxyolean-12-ene-23α-oic acid, 28-O-β-D-glucopyranosyl-(1 → 6)-β-D-glycosyl ester (2) by chemical and extensive spectroscopic methods including IR, 1D, 2D NMR and HRESIMS. Both of the saponins were evaluated for their cytotoxicities against HEK-293, A-549 and HeLa human cancer cells using the MTT method. All compounds showed no substantial cytotoxic activity against tested cell lines. However, dianosides M-N and the n-butanol fraction exhibited considerable haemolysis in human erythrocyte cells. The immunomodulatory properties of dianosides M-N were also evaluated in activated whole blood cells by PMA plus ionomycin. Dianosides M-N increased IL-1β concentration significantly whereas the n-butanol fraction slightly augmented IL-1β secretion. All compounds did not change IL-2 and IFN-γ levels considerably.

The interdomain flexible linker of the polypeptide GalNAc transferases dictates their long-range glycosylation preferences

DEFF Research Database (Denmark)

Rivas, Matilde De Las; Lira-Navarrete, Erandi; Daniel, Earnest James Paul

2017-01-01

The polypeptide GalNAc-transferases (GalNAc-Ts), that initiate mucin-type O-glycosylation, consist of a catalytic and a lectin domain connected by a flexible linker. In addition to recognizing polypeptide sequence, the GalNAc-Ts exhibit unique long-range N- A nd/or C-terminal prior glycosylation ...

Tumor-derived microvesicles mediate human breast cancer invasion through differentially glycosylated EMMPRIN.

Science.gov (United States)

Menck, Kerstin; Scharf, Christian; Bleckmann, Annalen; Dyck, Lydia; Rost, Ulrike; Wenzel, Dirk; Dhople, Vishnu M; Siam, Laila; Pukrop, Tobias; Binder, Claudia; Klemm, Florian

2015-04-01

Tumor cells secrete not only a variety of soluble factors, but also extracellular vesicles that are known to support the establishment of a favorable tumor niche by influencing the surrounding stroma cells. Here we show that tumor-derived microvesicles (T-MV) also directly influence the tumor cells by enhancing their invasion in a both autologous and heterologous manner. Neither the respective vesicle-free supernatant nor MV from benign mammary cells mediate invasion. Uptake of T-MV is essential for the proinvasive effect. We further identify the highly glycosylated form of the extracellular matrix metalloproteinase inducer (EMMPRIN) as a marker for proinvasive MV. EMMPRIN is also present at high levels on MV from metastatic breast cancer patients in vivo. Anti-EMMPRIN strategies, such as MV deglycosylation, gene knockdown, and specific blocking peptides, inhibit MV-induced invasion. Interestingly, the effect of EMMPRIN-bearing MV is not mediated by matrix metalloproteinases but by activation of the p38/MAPK signaling pathway in the tumor cells. In conclusion, T-MV stimulate cancer cell invasion via a direct feedback mechanism dependent on highly glycosylated EMMPRIN. © The Author (2014). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS.

Palladium- and copper-mediated N-aryl bond formation reactions for the synthesis of biological active compounds

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Burkhard Koenig

2011-01-01

Full Text Available N-Arylated aliphatic and aromatic amines are important substituents in many biologically active compounds. In the last few years, transition-metal-mediated N-aryl bond formation has become a standard procedure for the introduction of amines into aromatic systems. While N-arylation of simple aromatic halides by simple amines works with many of the described methods in high yield, the reactions may require detailed optimization if applied to the synthesis of complex molecules with additional functional groups, such as natural products or drugs. We discuss and compare in this review the three main N-arylation methods in their application to the synthesis of biologically active compounds: Palladium-catalysed Buchwald–Hartwig-type reactions, copper-mediated Ullmann-type and Chan–Lam-type N-arylation reactions. The discussed examples show that palladium-catalysed reactions are favoured for large-scale applications and tolerate sterically demanding substituents on the coupling partners better than Chan–Lam reactions. Chan–Lam N-arylations are particularly mild and do not require additional ligands, which facilitates the work-up. However, reaction times can be very long. Ullmann- and Buchwald–Hartwig-type methods have been used in intramolecular reactions, giving access to complex ring structures. All three N-arylation methods have specific advantages and disadvantages that should be considered when selecting the reaction conditions for a desired C–N bond formation in the course of a total synthesis or drug synthesis.

Glycosylation: a hallmark of cancer?

Science.gov (United States)

Vajaria, Bhairavi N; Patel, Prabhudas S

2017-04-01

The hallmarks of cancer are characterized by functional capabilities that allow cancer cells to survive, proliferate and disseminate during the multistep tumorigenesis. Cancer being a cellular disease, changes in cellular glycoproteins play an important role in malignant transformation and cancer progression. The present review summarizes various studies that depicted correlation of glycosylation with tumor initiation, progression and metastasis, which are helpful in early diagnosis, disease monitoring and prognosis. The results are further strengthened by our reports, which depicted alterations in sialylation and fucosylation in different cancers. Alterations in glycosyltransferases are also involved in formation of various tumor antigens (e.g. Sialyl Lewis x) which serves as ligand for the cell adhesion molecule, selectin which is involved in adhesion of cancer cells to vascular endothelium and thus contributes to hematogenous metastasis. Increased glycosylation accompanied by alterations in glycosyltranferases, glycosidases, glycans and mucins (MUC)s are also involved in loss of E-cadherin, a key molecule implicated in metastatic dissemination of cells. The present review also summarizes the correlation of glycosylation with all the hallmarks of cancer. The enormous progress in the design of novel inhibitors of pathway intermediates of sialylation and fucosylation can prove wonders in combating the dreadful disease. The results provide the evidence that altered glycosylation is linked to tumor initiation, progression and metastasis. Hence, it can be considered as a new hallmark of cancer development and strategies to develop novel glycosylation targeted molecules should be strengthened.

Evidence for Differential Glycosylation of Trophoblast Cell Types.

Science.gov (United States)

Chen, Qiushi; Pang, Poh-Choo; Cohen, Marie E; Longtine, Mark S; Schust, Danny J; Haslam, Stuart M; Blois, Sandra M; Dell, Anne; Clark, Gary F

2016-06-01

Human placental villi are surfaced by the syncytiotrophoblast (STB), with a layer of cytotrophoblasts (CTB) positioned just beneath the STB. STB in normal term pregnancies is exposed to maternal immune cells in the placental intervillous space. Extravillous cytotrophoblasts (EVT) invade the decidua and spiral arteries, where they act in conjunction with natural killer (NK) cells to convert the spiral arteries into flaccid conduits for maternal blood that support a 3-4 fold increase in the rate of maternal blood flow into the placental intervillous space. The functional roles of these distinct trophoblast subtypes during pregnancy suggested that they could be differentially glycosylated. Glycomic analysis of these trophoblasts has revealed the expression of elevated levels of biantennary N-glycans in STB and CTB, with the majority of them bearing a bisecting GlcNAc. N-glycans terminated with polylactosamine extensions were also detected at low levels. A subset of the N-glycans linked to these trophoblasts were sialylated, primarily with terminal NeuAcα2-3Gal sequences. EVT were decorated with the same N-glycans as STB and CTB, except in different proportions. The level of bisecting type N-glycans was reduced, but the level of N-glycans decorated with polylactosamine sequences were substantially elevated compared with the other types of trophoblasts. The level of triantennary and tetraantennary N-glycans was also elevated in EVT. The sialylated N-glycans derived from EVT were completely susceptible to an α2-3 specific neuraminidase (sialidase S). The possibility exists that the N-glycans associated with these different trophoblast subpopulations could act as functional groups. These potential relationships will be considered. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Fab glycosylation of immunoglobulin G does not associate with improvement of rheumatoid arthritis during pregnancy

NARCIS (Netherlands)

A. Bondt (Albert); M. Wuhrer (Manfred); T.M. Kuijper (Martijn); J.M.W. Hazes (Mieke); R.J.E.M. Dolhain (Radboud)

2016-01-01

textabstractBackground: Changes in immunoglobulin G (IgG) constant domain (Fc) glycosylation are associated with changes in rheumatoid arthritis (RA) disease activity in response to pregnancy. Here, we sought to determine whether the same holds true for variable domain (Fab) glycosylation. Methods:

Synthesis of N-(6-Arylbenzo[d]thiazole-2-acetamide Derivatives and Their Biological Activities: An Experimental and Computational Approach.

Science.gov (United States)

Gull, Yasmeen; Rasool, Nasir; Noreen, Mnaza; Altaf, Ataf Ali; Musharraf, Syed Ghulam; Zubair, Muhammad; Nasim, Faiz-Ul-Hassan; Yaqoob, Asma; DeFeo, Vincenzo; Zia-Ul-Haq, Muhammad

2016-02-25

A new series of N-(6-arylbenzo[d]thiazol-2-yl)acetamides were synthesized by C-C coupling methodology in the presence of Pd(0) using various aryl boronic pinacol ester/acids. The newly synthesized compounds were evaluated for various biological activities like antioxidant, haemolytic, antibacterial and urease inhibition. In bioassays these compounds were found to have moderate to good activities. Among the tested biological activities screened these compounds displayed the most significant activity for urease inhibition. In urease inhibition, all compounds were found more active than the standard used. The compound N-(6-(p-tolyl)benzo[d]thiazol-2-yl)acetamide was found to be the most active. To understand this urease inhibition, molecular docking studies were performed. The in silico studies showed that these acetamide derivatives bind to the non-metallic active site of the urease enzyme. Structure-activity studies revealed that H-bonding of compounds with the enzyme is important for its inhibition.

Isolation, N-glycosylations and Function of a Hyaluronidase-Like Enzyme from the Venom of the Spider Cupiennius salei.

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Olivier Biner

Full Text Available Hyaluronidases are important venom components acting as spreading factor of toxic compounds. In several studies this spreading effect was tested on vertebrate tissue. However, data about the spreading activity on invertebrates, the main prey organisms of spiders, are lacking. Here, a hyaluronidase-like enzyme was isolated from the venom of the spider Cupiennius salei. The amino acid sequence of the enzyme was determined by cDNA analysis of the venom gland transcriptome and confirmed by protein analysis. Two complex N-linked glycans akin to honey bee hyaluronidase glycosylations, were identified by tandem mass spectrometry. A C-terminal EGF-like domain was identified in spider hyaluronidase using InterPro. The spider hyaluronidase-like enzyme showed maximal activity at acidic pH, between 40-60°C, and 0.2 M KCl. Divalent ions did not enhance HA degradation activity, indicating that they are not recruited for catalysis.Besides hyaluronan, the enzyme degrades chondroitin sulfate A, whereas heparan sulfate and dermatan sulfate are not affected. The end products of hyaluronan degradation are tetramers, whereas chondroitin sulfate A is mainly degraded to hexamers. Identification of terminal N-acetylglucosamine or N-acetylgalactosamine at the reducing end of the oligomers identified the enzyme as an endo-β-N-acetyl-D-hexosaminidase hydrolase. The spreading effect of the hyaluronidase-like enzyme on invertebrate tissue was studied by coinjection of the enzyme with the Cupiennius salei main neurotoxin CsTx-1 into Drosophila flies. The enzyme significantly enhances the neurotoxic activity of CsTx-1. Comparative substrate degradation tests with hyaluronan, chondroitin sulfate A, dermatan sulfate, and heparan sulfate with venoms from 39 spider species from 21 families identified some spider families (Atypidae, Eresidae, Araneidae and Nephilidae without activity of hyaluronidase-like enzymes. This is interpreted as a loss of this enzyme and fits quite well

Isolation, N-glycosylations and Function of a Hyaluronidase-Like Enzyme from the Venom of the Spider Cupiennius salei

Science.gov (United States)

Trachsel, Christian; Moser, Aline; Kopp, Lukas; Langenegger, Nicolas; Kämpfer, Urs; von Ballmoos, Christoph; Nentwig, Wolfgang; Schürch, Stefan; Schaller, Johann

2015-01-01

Structure of Cupiennius salei venom hyaluronidase Hyaluronidases are important venom components acting as spreading factor of toxic compounds. In several studies this spreading effect was tested on vertebrate tissue. However, data about the spreading activity on invertebrates, the main prey organisms of spiders, are lacking. Here, a hyaluronidase-like enzyme was isolated from the venom of the spider Cupiennius salei. The amino acid sequence of the enzyme was determined by cDNA analysis of the venom gland transcriptome and confirmed by protein analysis. Two complex N-linked glycans akin to honey bee hyaluronidase glycosylations, were identified by tandem mass spectrometry. A C-terminal EGF-like domain was identified in spider hyaluronidase using InterPro. The spider hyaluronidase-like enzyme showed maximal activity at acidic pH, between 40–60°C, and 0.2 M KCl. Divalent ions did not enhance HA degradation activity, indicating that they are not recruited for catalysis. Function of venom hyaluronidases Besides hyaluronan, the enzyme degrades chondroitin sulfate A, whereas heparan sulfate and dermatan sulfate are not affected. The end products of hyaluronan degradation are tetramers, whereas chondroitin sulfate A is mainly degraded to hexamers. Identification of terminal N-acetylglucosamine or N-acetylgalactosamine at the reducing end of the oligomers identified the enzyme as an endo-β-N-acetyl-D-hexosaminidase hydrolase. The spreading effect of the hyaluronidase-like enzyme on invertebrate tissue was studied by coinjection of the enzyme with the Cupiennius salei main neurotoxin CsTx-1 into Drosophila flies. The enzyme significantly enhances the neurotoxic activity of CsTx-1. Comparative substrate degradation tests with hyaluronan, chondroitin sulfate A, dermatan sulfate, and heparan sulfate with venoms from 39 spider species from 21 families identified some spider families (Atypidae, Eresidae, Araneidae and Nephilidae) without activity of hyaluronidase-like enzymes

A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their glycosylation sites using HILIC enrichment and partial deglycosylation

DEFF Research Database (Denmark)

Hägglund, Per; Bunkenborg, Jakob; Elortza, Felix

2004-01-01

remains linked to the asparagine residue. The removal of the major part of the glycan simplifies the MS/MS fragment ion spectra of glycopeptides, while the remaining GlcNAc residue enables unambiguous assignment of the glycosylation site together with the amino acid sequence. We first tested our approach...

EDEM2 and OS-9 are required for ER-associated degradation of non-glycosylated sonic hedgehog.

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Hsiang-Yun Tang

Full Text Available Misfolded proteins of the endoplasmic reticulum (ER are eliminated by the ER-associated degradation (ERAD in eukaryotes. In S. cerevisiae, ER-resident lectins mediate substrate recognition through bipartite signals consisting of an unfolded local structure and the adjacent glycan. Trimming of the glycan is essential for the directional delivery of the substrates. Whether a similar recognition and delivery mechanism exists in mammalian cells is unknown. In this study, we systematically study the function and substrate specificity of known mammalian ER lectins, including EDEM1/2/3, OS-9 and XTP-3B using the recently identified ERAD substrate sonic hedgehog (SHH, a soluble protein carrying a single N-glycan, as well as its nonglycosylated mutant N278A. Efficient ERAD of N278A requires the core processing complex of HRD1, SEL1L and p97, similar to the glycosylated SHH. While EDEM2 was required for ERAD of both glycosylated and non-glycosylated SHHs, EDEM3 was only necessary for glycosylated SHH and EDEM1 was dispensable for both. Degradation of SHH and N278A also required OS-9, but not the related lectin XTP3-B. Robust interaction of both EDEM2 and OS-9 with a non-glycosylated SHH variant indicates that the misfolded polypeptide backbone, rather than a glycan signature, functions as the predominant signal for recognition for ERAD. Notably, SHH-N278A is the first nonglycosylated substrate to require EDEM2 for recognition and targeting for ERAD. EDEM2 also interacts with calnexin and SEL1L, suggesting a potential avenue by which misfolded glycoproteins may be shunted towards SEL1L and ERAD rather than being released into the secretory pathway. Thus, ER lectins participate in the recognition and delivery of misfolded ER substrates differently in mammals, with an underlying mechanism distinct from that of S. cerevisiae.

Implications of cellobiohydrolase glycosylation for use in biomass conversion

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Decker Stephen R

2008-05-01

Full Text Available Abstract The cellulase producing ascomycete, Trichoderma reesei (Hypocrea jecorina, is known to secrete a range of enzymes important for ethanol production from lignocellulosic biomass. It is also widely used for the commercial scale production of industrial enzymes because of its ability to produce high titers of heterologous proteins. During the secretion process, a number of post-translational events can occur, however, that impact protein function and stability. Another ascomycete, Aspergillus niger var. awamori, is also known to produce large quantities of heterologous proteins for industry. In this study, T. reesei Cel7A, a cellobiohydrolase, was expressed in A. niger var. awamori and subjected to detailed biophysical characterization. The purified recombinant enzyme contains six times the amount of N-linked glycan than the enzyme purified from a commercial T. reesei enzyme preparation. The activities of the two enzyme forms were compared using bacterial (microcrystalline and phosphoric acid swollen (amorphous cellulose as substrates. This comparison suggested that the increased level of N-glycosylation of the recombinant Cel7A (rCel7A resulted in reduced activity and increased non-productive binding on cellulose. When treated with the N-glycosidase PNGaseF, the molecular weight of the recombinant enzyme approached that of the commercial enzyme and the activity on cellulose was improved.

Glycosylation Alters Dimerization Properties of a Cell-surface Signaling Protein, Carcinoembryonic Antigen-related Cell Adhesion Molecule 1 (CEACAM1)*

Science.gov (United States)

Zhuo, You; Yang, Jeong-Yeh; Moremen, Kelley W.; Prestegard, James H.

2016-01-01

Human carcinoembryonic antigen-related cell adhesion molecule 1 (C?/Au: EACAM1) is a cell-surface signaling molecule involved in cell adhesion, proliferation, and immune response. It is also implicated in cancer angiogenesis, progression, and metastasis. This diverse set of effects likely arises as a result of the numerous homophilic and heterophilic interactions that CEACAM1 can have with itself and other molecules. Its N-terminal Ig variable (IgV) domain has been suggested to be a principal player in these interactions. Previous crystal structures of the β-sandwich-like IgV domain have been produced using Escherichia coli-expressed material, which lacks native glycosylation. These have led to distinctly different proposals for dimer interfaces, one involving interactions of ABED β-strands and the other involving GFCC′C″ β-strands, with the former burying one prominent glycosylation site. These structures raise questions as to which form may exist in solution and what the effect of glycosylation may have on this form. Here, we use NMR cross-correlation measurements to examine the effect of glycosylation on CEACAM1-IgV dimerization and use residual dipolar coupling (RDC) measurements to characterize the solution structure of the non-glycosylated form. Our findings demonstrate that even addition of a single N-linked GlcNAc at potential glycosylation sites inhibits dimer formation. Surprisingly, RDC data collected on E. coli expressed material in solution indicate that a dimer using the non-glycosylated GFCC′C″ interface is preferred even in the absence of glycosylation. The results open new questions about what other factors may facilitate dimerization of CEACAM1 in vivo, and what roles glycosylation may play in heterophylic interactions. PMID:27471271

Quinoxaline 1, 4-di-N-oxides: Biological activities and mechanisms of actions

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Guyue eCheng

2016-03-01

Full Text Available Quinoxaline 1, 4-di-N-oxides (QdNOs have manifold biological properties, including antimicrobial, antitumoral, antitrypanosomal and antiinflammatory/antioxidant activities. These diverse activities endow them broad applications and prospects in human and veterinary medicines. As QdNOs arouse widespread interest, the evaluation of their medicinal chemistry is still in progress. In the meantime, adverse effects have been reported in some of the QdNO derivatives. For example, genotoxicity and bacterial resistance have been found in QdNO antibacterial growth promoters, conferring urgent need for discovery of new QdNO drugs. However, the modes of actions of QdNOs are not fully understood, hindering the development and innovation of these promising compounds. Here, QdNOs are categorized based on the activities and usages, among which the antimicrobial activities are consist of antibacterial, antimycobacterial and anticandida activities, and the antiprotozoal activities include antitrypanosomal, antimalarial, antitrichomonas and antiamoebic activities. The structure-activity relationship and the mode of actions of each type of activity of QdNOs are summarized, and the toxicity and the underlying mechanisms are also discussed, providing insight for the future research and development of these fascinating compounds.

Glycosylation status of vitamin D binding protein in cancer patients.

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Rehder, Douglas S; Nelson, Randall W; Borges, Chad R

2009-10-01

On the basis of the results of activity studies, previous reports have suggested that vitamin D binding protein (DBP) is significantly or even completely deglycosylated in cancer patients, eliminating the molecular precursor of the immunologically important Gc macrophage activating factor (GcMAF), a glycosidase-derived product of DBP. The purpose of this investigation was to directly determine the relative degree of O-linked trisaccharide glycosylation of serum-derived DBP in human breast, colorectal, pancreatic, and prostate cancer patients. Results obtained by electrospray ionization-based mass spectrometric immunoassay showed that there was no significant depletion of DBP trisaccharide glycosylation in the 56 cancer patients examined relative to healthy controls. These results suggest that alternative hypotheses regarding the molecular and/or structural origins of GcMAF must be considered to explain the relative inability of cancer patient serum to activate macrophages.

Production, crystallization and X-ray characterization of chemically glycosylated hen egg-white lysozyme

International Nuclear Information System (INIS)

López-Jaramillo, F. J.; Pérez-Banderas, F.; Hernández-Mateo, F.; Santoyo-González, F.

2005-01-01

The feasibility of glycosylation post-purification has been demonstrated by introducing glucose into the model protein lysozyme via a novel reaction that is compatible with biological samples. The crystallization of glycoproteins is one of the challenges to be confronted by the crystallographic community in the frame of what is known as glycobiology. The state of the art for the crystallization of glycoproteins is not promising and removal of the carbohydrate chains is generally suggested since they are flexible and a source of heterogeneity. In this paper, the feasibility of introducing glucose into the model protein hen egg-white lysozyme via a post-purification glycosylation reaction that may turn any protein into a model glycoprotein whose carbohydrate fraction can be manipulated is demonstrated

Temporal expression of HIV-1 envelope proteins in baculovirus-infected insect cells: Implications for glycosylation and CD4 binding

International Nuclear Information System (INIS)

Murphy, C.I.; Lennick, M.; Lehar, S.M.; Beltz, G.A.; Young, E.

1990-01-01

Three different human immunodeficiency virus type I (HIV-1) envelope derived recombinant proteins and the full length human CD4 polypeptide were expressed in Spodoptera frugiperda (Sf9) cells. DNA constructs encoding CD4, gp120, gp160, and gp160 delta were cloned into the baculovirus expression vector pVL941 or a derivative and used to generate recombinant viruses in a cotransfection with DNA from Autographa californica nuclear polyhedrosis virus (AcMNPV). Western blotting of cell extracts of the recombinant HIV-1 proteins showed that for each construct two major bands specifically reacted with anti-HIV-1 envelope antiserum. These bands corresponded to glycosylated and nonglycosylated versions of the HIV proteins as determined by 3H-mannose labeling and tunicamycin treatment of infected cells. A time course of HIV envelope expression revealed that at early times post-infection (24 hours) the proteins were fully glycosylated and soluble in nonionic detergents. However, at later times postinfection (48 hours), expression levels of recombinant protein reached a maximum but most of the increase was due to a rise in the level of the nonglycosylated species, which was largely insoluble in nonionic detergents. Thus, it appears that Sf9 cells cannot process large amounts of glycosylated recombinant proteins efficiently. As a measure of biological activity, the CD4 binding ability of both glycosylated and nonglycosylated recombinant HIV envelope proteins was tested in a coimmunoprecipitation assay. The results showed that CD4 and the glycosylated versions of recombinant gp120 or gp160 delta specifically associated with one another in this analysis. Nonglycosylated gp120 or gp160 delta proteins from tunicamycin-treated cultures did immunoprecipitate with anti-HIV-1 antiserum but did not interact with CD4

A novel post-translational modification in nerve terminals: O-linked N-acetylglucosamine phosphorylation

DEFF Research Database (Denmark)

Graham, Mark E; Thaysen-Andersen, Morten; Bache, Nicolai

2011-01-01

Protein phosphorylation and glycosylation are the most common post-translational modifications observed in biology, frequently on the same protein. Assembly protein AP180 is a synapse-specific phosphoprotein and O-linked beta-N-acetylglucosamine (O-GlcNAc) modified glycoprotein. AP180 is involved......NAc-P to a Thr residue was confirmed by electron transfer dissociation MS. A second AP180 tryptic peptide was also glycosyl phosphorylated, but the site of modification was not assigned. Sequence similarities suggest there may be a common motif within AP180 involving glycosyl phosphorylation and dual flanking...... phosphorylation sites within 4 amino acid residues. This novel type of protein glycosyl phosphorylation adds a new signaling mechanism to the regulation of neurotransmission and more complexity to the study of O-GlcNAc modification....

Splitting of α-Helical Structure as Molecular Basis for Abolishing an Amyloid Formation by Multiple Glycosylation: A Molecular Dynamics Simulation Study

Energy Technology Data Exchange (ETDEWEB)

Choi, Youngjin [Hoseo University, Asan (Korea, Republic of); Cho, Eunae; Jung, Seunho [Konkuk University, Seoul (Korea, Republic of)

2016-07-15

Molecular details played by glycosylation are complicated by the subtle nature of variations in the glycan structure, and this complexity is one of the research barriers to establish structure-function relationship on the protein modification. This is particularly true for understanding the exact structural consequence of the glycosylation of the biological proteins. The present MD simulation revealed molecular-level mechanism of the glycosylation effect on the peptide to understand the experimentally observed phenomenon for inhibiting amyloid formation in the model peptide. The galactose residue on the Ser17 undermined the helical integrity of main protein region by enhancing sugar–amino acid interaction and perturbing natural interactions between amino acid residues.

Improving specific activity and thermostability of Escherichia coli phytase by structure-based rational design.

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Wu, Tzu-Hui; Chen, Chun-Chi; Cheng, Ya-Shan; Ko, Tzu-Ping; Lin, Cheng-Yen; Lai, Hui-Lin; Huang, Ting-Yung; Liu, Je-Ruei; Guo, Rey-Ting

2014-04-10

Escherichia coli phytase (EcAppA) which hydrolyzes phytate has been widely applied in the feed industry, but the need to improve the enzyme activity and thermostability remains. Here, we conduct rational design with two strategies to enhance the EcAppA performance. First, residues near the substrate binding pocket of EcAppA were modified according to the consensus sequence of two highly active Citrobacter phytases. One out of the eleven mutants, V89T, exhibited 17.5% increase in catalytic activity, which might be a result of stabilized protein folding. Second, the EcAppA glycosylation pattern was modified in accordance with the Citrobacter phytases. An N-glycosylation motif near the substrate binding site was disrupted to remove spatial hindrance for phytate entry and product departure. The de-glycosylated mutants showed 9.6% increase in specific activity. On the other hand, the EcAppA mutants that adopt N-glycosylation motifs from CbAppA showed improved thermostability that three mutants carrying single N-glycosylation motif exhibited 5.6-9.5% residual activity after treatment at 80°C (1.8% for wild type). Furthermore, the mutant carrying all three glycosylation motifs exhibited 27% residual activity. In conclusion, a successful rational design was performed to obtain several useful EcAppA mutants with better properties for further applications. Copyright © 2014 Elsevier B.V. All rights reserved.

Structural and immunological characterization of the N-glycans from the major yellow jacket allergen Ves v 2: The N-glycan structures are needed for the human antibody recognition

DEFF Research Database (Denmark)

Seppälä, Ulla; Selby, David; Monsalve, Rafael

2009-01-01

of the study was to characterize the glycosylation patterns in Ves v 2 isoallergens and to assess their immunological properties regarding antibody binding and T cell activation. The glycosylation sites and the carbohydrate structures were verified by use of tandem mass spectrometry (MS/MS). The immunological....... Non-glycosylated rVes v 2, however, induced T cell and cytokine responses comparable to glycosylated nVes v 2. The present study shows that N-glycan structures are needed for the antibody recognition but not for the T cell reactivity of Ves v 2 in vitro. The occurrences of carbohydrate......-specific antibodies against nVes v 2, however, suggest that non-mammalian glycan structures as in nVes v 2 may provide a link between T cells and other effector cells in allergic responses....

Predominant Expression of Hybrid N-Glycans Has Distinct Cellular Roles Relative to Complex and Oligomannose N-Glycans

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M. Kristen Hall

2016-06-01

Full Text Available Glycosylation modulates growth, maintenance, and stress signaling processes. Consequently, altered N-glycosylation is associated with reduced fitness and disease. Therefore, expanding our understanding of N-glycans in altering biological processes is of utmost interest. Herein, clustered regularly interspaced short palindromic repeats/caspase9 (CRISPR/Cas9 technology was employed to engineer a glycosylation mutant Chinese Hamster Ovary (CHO cell line, K16, which expresses predominantly hybrid type N-glycans. This newly engineered cell line enabled us to compare N-glycan effects on cellular properties of hybrid type N-glycans, to the well-established Pro−5 and Lec1 cell lines, which express complex and oligomannose types of N-glycans, respectively. Lectin binding studies revealed the predominant N-glycan expressed in K16 is hybrid type. Cell dissociation and migration assays demonstrated the greatest strength of cell–cell adhesion and fastest migratory rates for oligomannose N-glycans, and these properties decreased as oligomannose type were converted to hybrid type, and further decreased upon conversion to complex type. Next, we examined the roles of three general types of N-glycans on ectopic expression of E-cadherin, a cell–cell adhesion protein. Microscopy revealed more functional E-cadherin at the cell–cell border when N-glycans were oligomannose and these levels decreased as the oligomannose N-glycans were processed to hybrid and then to complex. Thus, we provide evidence that all three general types of N-glycans impact plasma membrane architecture and cellular properties.

2,4-dimethoxybenzyl: An amide protecting group for 2-acetamido glycosyl donors

DEFF Research Database (Denmark)

Kelly, N.M.; Jensen, Knud Jørgen

2001-01-01

2,4-Dimethoxybenzyl (Dmob) was used as an amide protecting group for 2-acetamido glycosyl donors. The N-Dmob group was introduced by imine formation between 2,4-dimethoxybenzaldehyde and d-glucosamine, followed by per-O-acylation, reduction to form the amine, and finally N-acetylation to give 1...

UGT74AN1, a Permissive Glycosyltransferase from Asclepias curassavica for the Regiospecific Steroid 3-O-Glycosylation.

Science.gov (United States)

Wen, Chao; Huang, Wei; Zhu, Xue-Lin; Li, Xiao-San; Zhang, Fan; Jiang, Ren-Wang

2018-02-02

A permissive steroid glycosyltransferase (UGT74AN1) from Asclepias curassavica exhibited robust capabilities for the regiospecific C3 glycosylation of cardiotonic steroids and C 21 steroid precursors, and unprecedented promiscuity toward 53 structurally diverse natural and unnatural compounds to form O-, N-, and S-glycosides, along with the catalytic reversibility for a one-pot transglycosylation reaction. These findings highlight UGT74AN1 as the first regiospecific catalyst for cardiotonic steroid C3 glycosylation and exhibit significant potential for glycosylation of diverse bioactive molecules in drug discovery.

Identification of a protein glycosylation operon from Campylobacter jejuni JCM 2013 and its heterologous expression in Escherichia coli.

Science.gov (United States)

Srichaisupakit, Akkaraphol; Ohashi, Takao; Fujiyama, Kazuhito

2014-09-01

Campylobacter jejuni is a human enteropathogenic bacterium possessing an N-glycosylation system. In this work, a protein glycosylation (pgl) operon conferring prokaryotic N-glycosylation in C. jejuni JCM 2013 was cloned and identified. Fourteen open reading frames (ORFs) were found in the pgl operon. The operon organization was similar to that of C. jejuni NCTC 11168, with 98% and 99% identities in overall nucleotide sequence and amino acid sequence, respectively. The pgl operon was heterologously co-expressed with model protein CmeA in the Escherichia coli BL21 ΔwaaL mutant. The immuno- and lectin-blotting analysis indicated the protein glycosylation on the recombinant CmeA. In addition, to analyze the glycan composition, the recombinant CmeA was purified and subjected to in-gel trypsin digestion followed by mass spectrometry analysis. The mass spectrometry analysis showed the presence of the N-acetylhexosamine residue at the reducing end but not the predicted di-N-acetylbacillosamine (diNAcBac) residue. Further glycan structural study using the conventional fluorophore-labeling method revealed the GalNAcα-GalNAcα-(Hex-)HexNAc-HexNAc-HexNAc-HexNAc structure. Transcriptional analysis showed that UDP-diNAcBac synthases and diNAcBac transferase are transcribed but might not function in the constructed system. In conclusion, a pgl operon from C. jejuni JCM 2013 successfully functioned in E. coli, resulting in the observed prokaryotic glycosylation. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

A bioinformatics prediction approach towards analyzing the glycosylation, co-expression and interaction patterns of epithelial membrane antigen (EMA/MUC1)

International Nuclear Information System (INIS)

Kalra, Rajkumar S.; Wadhwa, Renu

2015-01-01

Epithelial membrane antigen (EMA or MUC1) is a heavily glycosylated, type I transmembrane glycoprotein commonly expressed by epithelial cells of duct organs. It has been shown to be aberrantly glycosylated in several diseases including cancer. Protein sequence based annotation and analysis of glycosylation profile of glycoproteins by robust computational and comprehensive algorithms provides possible insights to the mechanism(s) of anomalous glycosylation. In present report, by using a number of bioinformatics applications we studied EMA/MUC1 and explored its trans-membrane structural domain sequence that is widely subjected to glycosylation. Exploration of different extracellular motifs led to prediction of N and O-linked glycosylation target sites. Based on the putative O-linked target sites, glycosylated moieties and pathways were envisaged. Furthermore, Protein network analysis demonstrated physical interaction of EMA with a number of proteins and confirmed its functional involvement in cell growth and proliferation pathways. Gene Ontology analysis suggested an involvement of EMA in a number of functions including signal transduction, protein binding, processing and transport along with glycosylation. Thus, present study explored potential of bioinformatics prediction approach in analyzing glycosylation, co-expression and interaction patterns of EMA/MUC1 glycoprotein

A bioinformatics prediction approach towards analyzing the glycosylation, co-expression and interaction patterns of epithelial membrane antigen (EMA/MUC1)

Energy Technology Data Exchange (ETDEWEB)

Kalra, Rajkumar S., E-mail: renu-wadhwa@aist.go.jp; Wadhwa, Renu, E-mail: renu-wadhwa@aist.go.jp [Cell Proliferation Research Group and DBT-AIST International Laboratory for Advanced Biomedicine, National Institute of Advanced Industrial Science and Technology (AIST Central 4), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562 (Japan)

2015-02-27

Epithelial membrane antigen (EMA or MUC1) is a heavily glycosylated, type I transmembrane glycoprotein commonly expressed by epithelial cells of duct organs. It has been shown to be aberrantly glycosylated in several diseases including cancer. Protein sequence based annotation and analysis of glycosylation profile of glycoproteins by robust computational and comprehensive algorithms provides possible insights to the mechanism(s) of anomalous glycosylation. In present report, by using a number of bioinformatics applications we studied EMA/MUC1 and explored its trans-membrane structural domain sequence that is widely subjected to glycosylation. Exploration of different extracellular motifs led to prediction of N and O-linked glycosylation target sites. Based on the putative O-linked target sites, glycosylated moieties and pathways were envisaged. Furthermore, Protein network analysis demonstrated physical interaction of EMA with a number of proteins and confirmed its functional involvement in cell growth and proliferation pathways. Gene Ontology analysis suggested an involvement of EMA in a number of functions including signal transduction, protein binding, processing and transport along with glycosylation. Thus, present study explored potential of bioinformatics prediction approach in analyzing glycosylation, co-expression and interaction patterns of EMA/MUC1 glycoprotein.

Sensitive and comprehensive analysis of O-glycosylation in biotherapeutics: a case study of novel erythropoiesis stimulating protein.

Science.gov (United States)

Kim, Unyong; Oh, Myung Jin; Seo, Youngsuk; Jeon, Yinae; Eom, Joon-Ho; An, Hyun Joo

2017-09-01

Glycosylation of recombinant human erythropoietins (rhEPOs) is significantly associated with drug's quality and potency. Thus, comprehensive characterization of glycosylation is vital to assess the biotherapeutic quality and establish the equivalency of biosimilar rhEPOs. However, current glycan analysis mainly focuses on the N-glycans due to the absence of analytical tools to liberate O-glycans with high sensitivity. We developed selective and sensitive method to profile native O-glycans on rhEPOs. O-glycosylation on rhEPO including O-acetylation on a sialic acid was comprehensively characterized. Details such as O-glycan structure and O-acetyl-modification site were obtained from tandem MS. This method may be applied to QC and batch analysis of not only rhEPOs but also other biotherapeutics bearing multiple O-glycosylations.

Simultaneous Release and Labeling of O- and N-Glycans Allowing for Rapid Glycomic Analysis by Online LC-UV-ESI-MS/MS.

Science.gov (United States)

Wang, Chengjian; Lu, Yu; Han, Jianli; Jin, Wanjun; Li, Lingmei; Zhang, Ying; Song, Xuezheng; Huang, Linjuan; Wang, Zhongfu

2018-05-24

Most glycoproteins and biological protein samples undergo both O- and N-glycosylation, making characterization of their structures very complicated and time-consuming. Nevertheless, to fully understand the biological functions of glycosylation, both the glycosylation forms need to be analyzed. Herein we report a versatile, convenient one-pot method in which O- and N-glycans are simultaneously released from glycoproteins and chromogenically labeled in situ and thus available for further characterization. In this procedure, glycoproteins are incubated with 1-phenyl-3-methyl-5-pyrazolone (PMP) in aqueous ammonium hydroxide, making O-glycans released from protein backbones by β-elimination and N-glycans liberated by alkaline hydrolysis. The released glycans are promptly derivatized with PMP in situ by Knoevenagel condensation and Michael addition, with peeling degradation almost completely prevented. The recovered mixture of O- and N-glycans as bis-PMP derivatives features strong ultraviolet (UV) absorbing ability and hydrophobicity, allowing for high-resolution chromatographic separation and high-sensitivity spectrometric detection. Using this technique, O- and N-glycans were simultaneously prepared from some model glycoproteins and complex biological samples, without significant peeling, desialylation, deacetylation, desulfation or other side-reactions, and then comprehensively analyzed by online HILIC-UV-ESI-MS/MS and RP-HPLC-UV-ESI-MS/MS, with which some novel O- and N-glycan structures were first found. This method provides a simple, versatile strategy for high-throughput glycomics analysis.

GtfA and GtfB Are Both Required for Protein O-Glycosylation in Lactobacillus plantarum

Science.gov (United States)

Lee, I-Chiao; van Swam, Iris I.; Tomita, Satoru; Morsomme, Pierre; Rolain, Thomas; Hols, Pascal; Bron, Peter A.

2014-01-01

Acm2, the major autolysin of Lactobacillus plantarum WCFS1, was recently found to be O-glycosylated with N-acetylhexosamine, likely N-acetylglucosamine (GlcNAc). In this study, we set out to identify the glycosylation machinery by employing a comparative genomics approach to identify Gtf1 homologues, which are involved in fimbria-associated protein 1 (Fap1) glycosylation in Streptococcus parasanguinis. This in silico approach resulted in the identification of 6 candidate L. plantarum WCFS1 genes with significant homology to Gtf1, namely, tagE1 to tagE6. These candidate genes were targeted by systematic gene deletion, followed by assessment of the consequences on glycosylation of Acm2. We observed a changed mobility of Acm2 on SDS-PAGE in the tagE5E6 deletion strain, while deletion of other tagE genes resulted in Acm2 mobility comparable to that of the wild type. Subsequent mass spectrometry analysis of excised and in-gel-digested Acm2 confirmed the loss of glycosylation on Acm2 in the tagE5E6 deletion mutant, whereas a lectin blot using GlcNAc-specific succinylated wheat germ agglutinin (sWGA) revealed that besides Acm2, tagE5E6 deletion also abolished all but one other sWGA-reactive, protease-sensitive signal. Only complementation of both tagE5 and tagE6 restored those sWGA lectin signals, establishing that TagE5 and TagE6 are both required for the glycosylation of Acm2 as well as the vast majority of other sWGA-reactive proteins. Finally, sWGA lectin blotting experiments using a panel of 8 other L. plantarum strains revealed that protein glycosylation is a common feature in L. plantarum strains. With the establishment of these enzymes as protein glycosyltransferases, we propose to rename TagE5 and TagE6 as GtfA and GtfB, respectively. PMID:24532775

Preparation, crystallization and preliminary X-ray diffraction studies of the glycosylated form of human interleukin-23

International Nuclear Information System (INIS)

Shirouzono, Takumi; Chirifu, Mami; Nakamura, Chiharu; Yamagata, Yuriko; Ikemizu, Shinji

2012-01-01

Interleukin-23 (IL-23), a member of the IL-12 family, is a heterodimeric cytokine composed of p19 and p40 subunits. Human p19 and p40 subunits were cloned and coexpressed in N-acetylglucosaminyltransferase I-negative 293S cells. The glycosylated human IL-23 was purified and crystallized by the hanging-drop vapour-diffusion method. Interleukin-23 (IL-23), a member of the IL-12 family, is a heterodimeric cytokine composed of p19 and p40 subunits. IL-23 plays crucial roles in the activation, proliferation and survival of IL-17-producing helper T cells which induce various autoimmune diseases. Human p19 and p40 subunits were cloned and coexpressed in N-acetylglucosaminyltransferase I-negative 293S cells, which produce high-mannose-type glycosylated proteins in order to diminish the heterogeneity of modified N-linked glycans. The glycosylated human IL-23 was purified and crystallized by the hanging-drop vapour-diffusion method. X-ray diffraction data were then collected to 2.6 Å resolution. The crystal belonged to space group P6 1 or P6 5 , with unit-cell parameters a = b = 108.94, c = 83.79 Å, γ = 120°. Assuming that the crystal contains one molecule per asymmetric unit, the calculated Matthews coefficient was 2.69 Å 3 Da −1 , with a solvent content of 54.2%. The structure was determined by the molecular-replacement method, with an initial R factor of 52.6%. After subsequent rigid-body and positional refinement, the R work and R free values decreased to 31.4% and 38.7%, respectively

Rapid and individual-specific glycoprofiling of the low abundance N-glycosylated protein tissue inhibitor of metalloproteinases-1

DEFF Research Database (Denmark)

Thaysen-Andersen, Morten; Thøgersen, Ida B.; Nielsen, Hans Jørgen

2007-01-01

A gel-based method for a mass spectrometric site-specific glycoanalysis was developed using a recombinant glycoprotein expressed in two different cell lines. Hydrophilic interaction liquid chromatography at nanoscale level was used to enrich for glycopeptides prior to MS. The glycoprofiling...... glycoprofiling of a low abundance glycoprotein performed in an individual-specific manner allows for future studies of glycosylated biomarkers for person-specific detection of altered glycosylation and may thus allow early detection and monitoring of diseases....

High-throughput analysis of endogenous fruit glycosyl hydrolases using a novel chromogenic hydrogel substrate assay

DEFF Research Database (Denmark)

Schückel, Julia; Kracun, Stjepan Kresimir; Lausen, Thomas Frederik

2017-01-01

A broad range of enzyme activities can be found in a wide range of different fruits and fruiting bodies but there is a lack of methods where many samples can be handled in a high-throughput and efficient manner. In particular, plant polysaccharide degrading enzymes – glycosyl hydrolases (GHs) play...... led to a more profound understanding of the importance of GH activity and regulation, current methods for determining glycosyl hydrolase activity are lacking in throughput and fail to keep up with data output from transcriptome research. Here we present the use of a versatile, easy...

Secretion of biologically active glycoforms of bovine follicle stimulating hormone in plants

NARCIS (Netherlands)

Dirnberger, D.; Steinkellner, H.; Abdennebi, L.; Remy, J.J.; Wiel, van de D.

2001-01-01

We chose the follicle stimulating hormone (FSH), a pituitary heterodimeric glycoprotein hormone, as a model to assess the ability of the plant cell to express a recombinant protein that requires extensive N-glycosylation for subunit folding and assembly, intracellular trafficking, signal

Synthesis of Curcumin Glycosides with Enhanced Anticancer Properties Using One-Pot Multienzyme Glycosylation Technique.

Science.gov (United States)

Gurung, Rit Bahadur; Gong, So Youn; Dhakal, Dipesh; Le, Tuoi Thi; Jung, Na Rae; Jung, Hye Jin; Oh, Tae Jin; Sohng, Jae Kyung

2017-09-28

Curcumin is a natural polyphenolic compound, widely acclaimed for its antioxidant, antiinflammatory, antibacterial, and anticancerous properties. However, its use has been limited due to its low-aqueous solubility and poor bioavailability, rapid clearance, and low cellular uptake. In order to assess the effect of glycosylation on the pharmacological properties of curcumin, one-pot multienzyme (OPME) chemoenzymatic glycosylation reactions with UDP- α-D-glucose or UDP-α-D-2-deoxyglucose as donor substrate were employed. The result indicated significant conversion of curcumin to its glycosylated derivatives: curcumin 4'- O -β- glucoside, curcumin 4',4''-di- O -β-glucoside, curcumin 4'- O -β-2-deoxyglucoside, and curcumin 4',4''-di- O -β-2-deoxyglucoside. The products were characterized by ultra-fast performance liquid chromatography, high-resolution quadruple-time-of-flight electrospray ionization-mass spectrometry, and NMR analyses. All the products showed improved water solubility and comparable antibacterial activities. Additionally, the curcumin 4'- O -β-glucoside and curcumin 4'- O -β-2-deoxyglucoside showed enhanced anticancer activities compared with the parent aglycone and diglycoside derivatives. This result indicates that glycosylation can be an effective approach for enhancing the pharmaceutical properties of different natural products, such as curcumin.

Immunoglobulin G (IgG) Fab Glycosylation Analysis Using a New Mass Spectrometric High-throughput Profiling Method Reveals Pregnancy-associated Changes*

Science.gov (United States)

Bondt, Albert; Rombouts, Yoann; Selman, Maurice H. J.; Hensbergen, Paul J.; Reiding, Karli R.; Hazes, Johanna M. W.; Dolhain, Radboud J. E. M.; Wuhrer, Manfred

2014-01-01

The N-linked glycosylation of the constant fragment (Fc) of immunoglobulin G has been shown to change during pathological and physiological events and to strongly influence antibody inflammatory properties. In contrast, little is known about Fab-linked N-glycosylation, carried by ∼20% of IgG. Here we present a high-throughput workflow to analyze Fab and Fc glycosylation of polyclonal IgG purified from 5 μl of serum. We were able to detect and quantify 37 different N-glycans by means of MALDI-TOF-MS analysis in reflectron positive mode using a novel linkage-specific derivatization of sialic acid. This method was applied to 174 samples of a pregnancy cohort to reveal Fab glycosylation features and their change with pregnancy. Data analysis revealed marked differences between Fab and Fc glycosylation, especially in the levels of galactosylation and sialylation, incidence of bisecting GlcNAc, and presence of high mannose structures, which were all higher in the Fab portion than the Fc, whereas Fc showed higher levels of fucosylation. Additionally, we observed several changes during pregnancy and after delivery. Fab N-glycan sialylation was increased and bisection was decreased relative to postpartum time points, and nearly complete galactosylation of Fab glycans was observed throughout. Fc glycosylation changes were similar to results described before, with increased galactosylation and sialylation and decreased bisection during pregnancy. We expect that the parallel analysis of IgG Fab and Fc, as set up in this paper, will be important for unraveling roles of these glycans in (auto)immunity, which may be mediated via recognition by human lectins or modulation of antigen binding. PMID:25004930

Osteoblasts extracellular matrix induces vessel like structures through glycosylated collagen I

Energy Technology Data Exchange (ETDEWEB)

Palmieri, D. [Genetics, DIBIO, University of Genova, Corso Europa 26, 16132 Genova (Italy); Valli, M.; Viglio, S. [Department of Biochemistry, University of Pavia (Italy); Ferrari, N. [Istituto Nazionale per la ricerca sul Cancro, Genova (Italy); Ledda, B.; Volta, C. [Genetics, DIBIO, University of Genova, Corso Europa 26, 16132 Genova (Italy); Manduca, P., E-mail: man-via@unige.it [Genetics, DIBIO, University of Genova, Corso Europa 26, 16132 Genova (Italy)

2010-03-10

Extracellular matrix (ECM) plays a fundamental role in angiogenesis affecting endothelial cells proliferation, migration and differentiation. Vessels-like network formation in vitro is a reliable test to study the inductive effects of ECM on angiogenesis. Here we utilized matrix deposed by osteoblasts as substrate where the molecular and structural complexity of the endogenous ECM is preserved, to test if it induces vessel-like network formation by endothelial cells in vitro. ECM is more similar to the physiological substrate in vivo than other substrates previously utilized for these studies in vitro. Osteogenic ECM, prepared in vitro from mature osteoblasts at the phase of maximal deposition and glycosylation of collagen I, induces EAhy926, HUVEC, and HDMEC endothelial cells to form vessels-like structures and promotes the activation of metalloproteinase-2 (MMP-2); the functionality of the p-38/MAPK signaling pathway is required. Osteogenic ECM also induces a transient increase of CXCL12 and a decrease of the receptor CXCR4. The induction of vessel-like networks is dependent from proper glycosylation of collagens and does not occur on osteogenic ECMs if deglycosylated by -galactosidase or on less glycosylated ECMs derived from preosteoblasts and normal fibroblasts, while is sustained on ECM from osteogenesis imperfecta fibroblasts only when their mutation is associated with over-glycosylation of collagen type I. These data support that post-translational glycosylation has a role in the induction in endothelial cells in vitro of molecules conductive to self-organization in vessels-like structures.

Osteoblasts extracellular matrix induces vessel like structures through glycosylated collagen I

International Nuclear Information System (INIS)

Palmieri, D.; Valli, M.; Viglio, S.; Ferrari, N.; Ledda, B.; Volta, C.; Manduca, P.

2010-01-01

Extracellular matrix (ECM) plays a fundamental role in angiogenesis affecting endothelial cells proliferation, migration and differentiation. Vessels-like network formation in vitro is a reliable test to study the inductive effects of ECM on angiogenesis. Here we utilized matrix deposed by osteoblasts as substrate where the molecular and structural complexity of the endogenous ECM is preserved, to test if it induces vessel-like network formation by endothelial cells in vitro. ECM is more similar to the physiological substrate in vivo than other substrates previously utilized for these studies in vitro. Osteogenic ECM, prepared in vitro from mature osteoblasts at the phase of maximal deposition and glycosylation of collagen I, induces EAhy926, HUVEC, and HDMEC endothelial cells to form vessels-like structures and promotes the activation of metalloproteinase-2 (MMP-2); the functionality of the p-38/MAPK signaling pathway is required. Osteogenic ECM also induces a transient increase of CXCL12 and a decrease of the receptor CXCR4. The induction of vessel-like networks is dependent from proper glycosylation of collagens and does not occur on osteogenic ECMs if deglycosylated by -galactosidase or on less glycosylated ECMs derived from preosteoblasts and normal fibroblasts, while is sustained on ECM from osteogenesis imperfecta fibroblasts only when their mutation is associated with over-glycosylation of collagen type I. These data support that post-translational glycosylation has a role in the induction in endothelial cells in vitro of molecules conductive to self-organization in vessels-like structures.

Identification of glycosylation sites in the SU component of the Avian Sarcoma/Leukosis virus Envelope Glycoprotein (Subgroup A) by mass spectrometry

International Nuclear Information System (INIS)

Kvaratskhelia, Mamuka; Clark, Patrick K.; Hess, Sonja; Melder, Deborah C.; Federspiel, Mark J.; Hughes, Stephen H.

2004-01-01

We used enzymatic digestion and mass spectrometry to identify the sites of glycosylation on the SU component of the Avian Sarcoma/Leukosis virus (ASLV) Envelope Glycoprotein (Subgroup A). The analysis was done with an SU(A)-rIgG fusion protein that binds the cognate receptor (Tva) specifically. PNGase F removed all the carbohydrate from the SU(A)-rIgG fusion. PNGase F is specific for N-linked carbohydrates; this shows that all the carbohydrate on SU(A) is N-linked. There are 10 modified aspargines in SU(A) (N17, N59, N80, N97, N117, N196, N230, N246, N254, and N330). All conform to the consensus site for N-linked glycosylation NXS/T. There is one potential glycosylation site (N236) that is not modified. Removing most of the carbohydrate from the mature SU(A)-rIgG by PNGase F treatment greatly reduces the ability of the protein to bind Tva, suggesting that carbohydrate may play a direct role in receptor binding

Glycosylation intermediates studied using low temperature ¹H- and ¹⁹F-DOSY NMR

DEFF Research Database (Denmark)

Qiao, Yan; Ge, Wenzhi; Jia, Lingyu

2016-01-01

Low temperature 1H- and 19F-DOSY have been used for analyzing reactive intermediates in glycosylation reactions, where a glycosyl trichloroacetimidate donor has been activated using different catalysts. The DOSY protocols have been optimized for low temperature experiments and provided new insight...

The glycosylation and characterization of the candidate Gc macrophage activating factor

DEFF Research Database (Denmark)

Ravnsborg, Tina; Olsen, Dorthe T; Thysen, Anna Hammerich

2010-01-01

The vitamin D binding protein, Gc globulin, has in recent years received some attention for its role as precursor for the extremely potent macrophage activating factor (GcMAF). An O-linked trisaccharide has been allocated to the threonine residue at position 420 in two of the three most common...... isoforms of Gc globulin (Gc1s and Gc1f). A substitution for a lysine residue at position 420 in Gc2 prevents this isoform from being glycosylated at that position. It has been suggested that Gc globulin subjected sequentially to sialidase and galactosidase treatment generates GcMAF in the form of Gc...... globulin with only a single GalNAc attached to T420. In this study we confirm the location of a linear trisaccharide on T420. Furthermore, we provide the first structural evidence of the generation of the proposed GcMAF by use of glycosidase treatment and mass spectrometry. Additionally the generated GcMAF...

Detection of site specific glycosylation in proteins using flow cytometry†

Science.gov (United States)

Jayakumar, Deepak; Marathe, Dhananjay D.; Neelamegham, Sriram

2009-01-01

We tested the possibility that it is possible to express unique peptide probes on cell surfaces and detect site-specific glycosylation on these peptides using flow cytometry. Such development can enhance the application of flow cytometry to detect and quantify post-translational modifications in proteins. To this end, the N-terminal section of the human leukocyte glycoprotein PSGL-1 (P-selectin glycoprotein ligand-1) was modified to contain a poly-histidine tag followed by a proteolytic cleavage site. Amino acids preceding the cleavage site have a single O-linked glycosylation site. The recombinant protein called PSGL-1 (HT) was expressed on the surface of two mammalian cell lines, CHO and HL-60, using a lentiviral delivery approach. Results demonstrate that the N-terminal portion of PSGL-1 (HT) can be released from these cells by protease, and the resulting peptide can be readily captured and detected using cytometry-bead assays. Using this strategy, the peptide was immunoprecipitated onto beads bearing mAbs against either the poly-histidine sequence or the human PSGL-1. The carbohydrate epitope associated with the released peptide was detected using HECA-452 and CSLEX-1, monoclonal antibodies that recognize the sialyl Lewis-X epitope. Finally, the peptide released from cells could be separated and enriched using nickel chelate beads. Overall, such an approach that combines recombinant protein expression with flow cytometry, may be useful to quantify changes in site-specific glycosylation for basic science and clinical applications. PMID:19735085

Halide-mediated regioselective 6-O-glycosylation of unprotected hexopyranosides with perbenzylated glycosyl bromide donors

DEFF Research Database (Denmark)

Niedbal, Dominika Alina; Madsen, Robert

2016-01-01

The regio- and stereoselective glycosylation at the 6-position in 2,3,4,6-unprotected hexopyranosides has been investigated with dibutyltin oxide as the directing agent. Perbenzylated hexopyranosyl bromides were employed as the donors and the glycosylations were promoted by tetrabutylammonium...... bromide. The couplings were completely selective for both glucose and galactose donors and acceptors as long as the stannylene acetal of the acceptor was soluble in dichloromethane. This gave rise to a number of 1,2-cis-linked disaccharides in reasonable yields. Mannose donors and acceptors, on the other...

Glycosylation of the self-recognizing Escherichia coli Ag43 autotransporter protein

DEFF Research Database (Denmark)

Sherlock, O.; Dobrindt, U.; Jensen, J.B.

2006-01-01

a novel member to this exclusive group, namely, antigen 43 (Ag43), a self-recognizing autotransporter protein. By mass spectrometry Ag43 was demonstrated to be glycosylated by addition of heptose residues at several positions in the passenger domain. Glycosylation of Ag43 by the action of the Aah and Tib......C glycosyltransferases was observed in laboratory strains. Importantly, Ag43 was also found to be glycosylated in a wild-type strain, suggesting that Ag43-glycosylation may be a widespread phenomenon. Glycosylation of Ag43 does not seem to interfere with its self-associating properties. However, the glycosylated form...

Overelaborated synaptic architecture and reduced synaptomatrix glycosylation in a Drosophila classic galactosemia disease model

Directory of Open Access Journals (Sweden)

Patricia Jumbo-Lucioni

2014-12-01

Full Text Available Classic galactosemia (CG is an autosomal recessive disorder resulting from loss of galactose-1-phosphate uridyltransferase (GALT, which catalyzes conversion of galactose-1-phosphate and uridine diphosphate (UDP-glucose to glucose-1-phosphate and UDP-galactose, immediately upstream of UDP–N-acetylgalactosamine and UDP–N-acetylglucosamine synthesis. These four UDP-sugars are essential donors for driving the synthesis of glycoproteins and glycolipids, which heavily decorate cell surfaces and extracellular spaces. In addition to acute, potentially lethal neonatal symptoms, maturing individuals with CG develop striking neurodevelopmental, motor and cognitive impairments. Previous studies suggest that neurological symptoms are associated with glycosylation defects, with CG recently being described as a congenital disorder of glycosylation (CDG, showing defects in both N- and O-linked glycans. Here, we characterize behavioral traits, synaptic development and glycosylated synaptomatrix formation in a GALT-deficient Drosophila disease model. Loss of Drosophila GALT (dGALT greatly impairs coordinated movement and results in structural overelaboration and architectural abnormalities at the neuromuscular junction (NMJ. Dietary galactose and mutation of galactokinase (dGALK or UDP-glucose dehydrogenase (sugarless genes are identified, respectively, as critical environmental and genetic modifiers of behavioral and cellular defects. Assaying the NMJ extracellular synaptomatrix with a broad panel of lectin probes reveals profound alterations in dGALT mutants, including depletion of galactosyl, N-acetylgalactosamine and fucosylated horseradish peroxidase (HRP moieties, which are differentially corrected by dGALK co-removal and sugarless overexpression. Synaptogenesis relies on trans-synaptic signals modulated by this synaptomatrix carbohydrate environment, and dGALT-null NMJs display striking changes in heparan sulfate proteoglycan (HSPG co-receptor and Wnt

Digestibility and IgE-Binding of Glycosylated Codfish Parvalbumin

Science.gov (United States)

de Jongh, Harmen H. J.; Robles, Carlos López; Nordlee, Julie A.; Lee, Poi-Wah; Baumert, Joseph L.; Hamilton, Robert G.; Taylor, Steve L.; Koppelman, Stef J.

2013-01-01

Food-processing conditions may alter the allergenicity of food proteins by different means. In this study, the effect of the glycosylation as a result of thermal treatment on the digestibility and IgE-binding of codfish parvalbumin is investigated. Native and glycosylated parvalbumins were digested with pepsin at various conditions relevant for the gastrointestinal tract. Intact proteins and peptides were analysed for apparent molecular weight and IgE-binding. Glycosylation did not substantially affect the digestion. Although the peptides resulting from digestion were relatively large (3 and 4 kDa), the IgE-binding was strongly diminished. However, the glycosylated parvalbumin had a strong propensity to form dimers and tetramers, and these multimers bound IgE intensely, suggesting stronger IgE-binding than monomeric parvalbumin. We conclude that glycosylation of codfish parvalbumin does not affect the digestibility of parvalbumin and that the peptides resulting from this digestion show low IgE-binding, regardless of glycosylation. Glycosylation of parvalbumin leads to the formation of higher order structures that are more potent IgE binders than native, monomeric parvalbumin. Therefore, food-processing conditions applied to fish allergen can potentially lead to increased allergenicity, even while the protein's digestibility is not affected by such processing. PMID:23878817

Digestibility and IgE-Binding of Glycosylated Codfish Parvalbumin

Directory of Open Access Journals (Sweden)

Harmen H. J. de Jongh

2013-01-01

Full Text Available Food-processing conditions may alter the allergenicity of food proteins by different means. In this study, the effect of the glycosylation as a result of thermal treatment on the digestibility and IgE-binding of codfish parvalbumin is investigated. Native and glycosylated parvalbumins were digested with pepsin at various conditions relevant for the gastrointestinal tract. Intact proteins and peptides were analysed for apparent molecular weight and IgE-binding. Glycosylation did not substantially affect the digestion. Although the peptides resulting from digestion were relatively large (3 and 4 kDa, the IgE-binding was strongly diminished. However, the glycosylated parvalbumin had a strong propensity to form dimers and tetramers, and these multimers bound IgE intensely, suggesting stronger IgE-binding than monomeric parvalbumin. We conclude that glycosylation of codfish parvalbumin does not affect the digestibility of parvalbumin and that the peptides resulting from this digestion show low IgE-binding, regardless of glycosylation. Glycosylation of parvalbumin leads to the formation of higher order structures that are more potent IgE binders than native, monomeric parvalbumin. Therefore, food-processing conditions applied to fish allergen can potentially lead to increased allergenicity, even while the protein’s digestibility is not affected by such processing.

High abundance of Serine/Threonine-rich regions predicted to be hyper-O-glycosylated in the secretory proteins coded by eight fungal genomes

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González Mario

2012-09-01

Full Text Available Abstract Background O-glycosylation of secretory proteins has been found to be an important factor in fungal biology and virulence. It consists in the addition of short glycosidic chains to Ser or Thr residues in the protein backbone via O-glycosidic bonds. Secretory proteins in fungi frequently display Ser/Thr rich regions that could be sites of extensive O-glycosylation. We have analyzed in silico the complete sets of putatively secretory proteins coded by eight fungal genomes (Botrytis cinerea, Magnaporthe grisea, Sclerotinia sclerotiorum, Ustilago maydis, Aspergillus nidulans, Neurospora crassa, Trichoderma reesei, and Saccharomyces cerevisiae in search of Ser/Thr-rich regions as well as regions predicted to be highly O-glycosylated by NetOGlyc (http://www.cbs.dtu.dk. Results By comparison with experimental data, NetOGlyc was found to overestimate the number of O-glycosylation sites in fungi by a factor of 1.5, but to be quite reliable in the prediction of highly O-glycosylated regions. About half of secretory proteins have at least one Ser/Thr-rich region, with a Ser/Thr content of at least 40% over an average length of 40 amino acids. Most secretory proteins in filamentous fungi were predicted to be O-glycosylated, sometimes in dozens or even hundreds of sites. Residues predicted to be O-glycosylated have a tendency to be grouped together forming hyper-O-glycosylated regions of varying length. Conclusions About one fourth of secretory fungal proteins were predicted to have at least one hyper-O-glycosylated region, which consists of 45 amino acids on average and displays at least one O-glycosylated Ser or Thr every four residues. These putative highly O-glycosylated regions can be found anywhere along the proteins but have a slight tendency to be at either one of the two ends.

Glycosyl-Nucleolipids as new bioinspired amphiphiles.

Science.gov (United States)

Latxague, Laurent; Patwa, Amit; Amigues, Eric; Barthélémy, Philippe

2013-09-30

Four new Glycosyl-NucleoLipid (GNL) analogs featuring either a single fluorocarbon or double hydrocarbon chains were synthesized in good yields from azido thymidine as starting material. Physicochemical studies (surface tension measurements, differential scanning calorimetry) indicate that hydroxybutanamide-based GNLs feature endothermic phase transition temperatures like the previously reported double chain glycerol-based GNLs. The second generation of GNFs featuring a free nucleobase reported here presents a better surface activity (lower glim) compared to the first generation of GNFs.

Synergizing metabolic flux analysis and nucleotide sugar metabolism to understand the control of glycosylation of recombinant protein in CHO cells

LENUS (Irish Health Repository)

Burleigh, Susan C

2011-10-18

Abstract Background The glycosylation of recombinant proteins can be altered by a range of parameters including cellular metabolism, metabolic flux and the efficiency of the glycosylation process. We present an experimental set-up that allows determination of these key processes associated with the control of N-linked glycosylation of recombinant proteins. Results Chinese hamster ovary cells (CHO) were cultivated in shake flasks at 0 mM glutamine and displayed a reduced growth rate, glucose metabolism and a slower decrease in pH, when compared to other glutamine-supplemented cultures. The N-linked glycosylation of recombinant human chorionic gonadotrophin (HCG) was also altered under these conditions; the sialylation, fucosylation and antennarity decreased, while the proportion of neutral structures increased. A continuous culture set-up was subsequently used to understand the control of HCG glycosylation in the presence of varied glutamine concentrations; when glycolytic flux was reduced in the absence of glutamine, the glycosylation changes that were observed in shake flask culture were similarly detected. The intracellular content of UDP-GlcNAc was also reduced, which correlated with a decrease in sialylation and antennarity of the N-linked glycans attached to HCG. Conclusions The use of metabolic flux analysis illustrated a case of steady state multiplicity, where use of the same operating conditions at each steady state resulted in altered flux through glycolysis and the TCA cycle. This study clearly demonstrated that the control of glycoprotein microheterogeneity may be examined by use of a continuous culture system, metabolic flux analysis and assay of intracellular nucleotides. This system advances our knowledge of the relationship between metabolic flux and the glycosylation of biotherapeutics in CHO cells and will be of benefit to the bioprocessing industry.

The role of glycosylation and domain interactions in the thermal stability of human angiotensin-converting enzyme.

Science.gov (United States)

O'Neill, Hester G; Redelinghuys, Pierre; Schwager, Sylva L U; Sturrock, Edward D

2008-09-01

The N and C domains of somatic angiotensin-converting enzyme (sACE) differ in terms of their substrate specificity, inhibitor profiling, chloride dependency and thermal stability. The C domain is thermally less stable than sACE or the N domain. Since both domains are heavily glycosylated, the effect of glycosylation on their thermal stability was investigated by assessing their catalytic and physicochemical properties. Testis ACE (tACE) expressed in mammalian cells, mammalian cells in the presence of a glucosidase inhibitor and insect cells yielded proteins with altered catalytic and physicochemical properties, indicating that the more complex glycans confer greater thermal stabilization. Furthermore, a decrease in tACE and N-domain N-glycans using site-directed mutagenesis decreased their thermal stability, suggesting that certain N-glycans have an important effect on the protein's thermodynamic properties. Evaluation of the thermal stability of sACE domain swopover and domain duplication mutants, together with sACE expressed in insect cells, showed that the C domain contained in sACE is less dependent on glycosylation for thermal stabilization than a single C domain, indicating that stabilizing interactions between the two domains contribute to the thermal stability of sACE and are decreased in a C-domain-duplicating mutant.

Does Glycosylation as a modifier of Original Antigenic Sin explain the case age distribution and unusual toxicity in pandemic novel H1N1 influenza?

Directory of Open Access Journals (Sweden)

Nishiura Hiroshi

2010-01-01

Full Text Available Abstract Background A pandemic novel H1N1 swine-origin influenza virus has emerged. Most recently the World Health Organization has announced that in a country-dependent fashion, up to 15% of cases may require hospitalization, often including respiratory support. It is now clear that healthy children and young adults are disproportionately affected, most unusually among those with severe respiratory disease without underlying conditions. One possible explanation for this case age distribution is the doctrine of Original Antigenic Sin, i.e., novel H1N1 may be antigenically similar to H1N1 viruses that circulated at an earlier time. Persons whose first exposure to influenza viruses was to such similar viruses would be relatively immune. However, this principle is not sufficient to explain the graded susceptibility between ages 20 and 60, the reduced susceptibility in children below age 10, and the unusual toxicity observed. Methods We collected case data from 11 countries, about 60% of all cases reported through mid-July 2009. We compared sequence data for the hemagglutinin of novel H1N1 with sequences of H1N1 viruses from 1918 to the present. We searched for sequence differences that imply loss of antigenicity either directly through amino acid substitution or by the appearance of sites for potential glycosylation proximal to sites known to be antigenic in humans. We also considered T-cell epitopes. Results In our composite, over 75% of confirmed cases of novel H1N1 occurred in persons ≤ 30 years old, with peak incidence in the age range 10-19 years. Less than 3% of cases occurred in persons over 65, with a gradation in incidence between ages 20 and 60 years. The sequence data indicates that novel H1N1 is most similar to H1N1 viruses that circulated before 1943. Novel H1N1 lacks glycosylation sites on the globular head of hemagglutinin (HA1 near antigenic regions, a pattern shared with the 1918 pandemic strain and H1N1 viruses that circulated

Channel sialic acids limit hERG channel activity during the ventricular action potential.

Science.gov (United States)

Norring, Sarah A; Ednie, Andrew R; Schwetz, Tara A; Du, Dongping; Yang, Hui; Bennett, Eric S

2013-02-01

Activity of human ether-a-go-go-related gene (hERG) 1 voltage-gated K(+) channels is responsible for portions of phase 2 and phase 3 repolarization of the human ventricular action potential. Here, we questioned whether and how physiologically and pathophysiologically relevant changes in surface N-glycosylation modified hERG channel function. Voltage-dependent hERG channel gating and activity were evaluated as expressed in a set of Chinese hamster ovary (CHO) cell lines under conditions of full glycosylation, no sialylation, no complex N-glycans, and following enzymatic deglycosylation of surface N-glycans. For each condition of reduced glycosylation, hERG channel steady-state activation and inactivation relationships were shifted linearly by significant depolarizing ∼9 and ∼18 mV, respectively. The hERG window current increased significantly by 50-150%, and the peak shifted by a depolarizing ∼10 mV. There was no significant change in maximum hERG current density. Deglycosylated channels were significantly more active (20-80%) than glycosylated controls during phases 2 and 3 of action potential clamp protocols. Simulations of hERG current and ventricular action potentials corroborated experimental data and predicted reduced sialylation leads to a 50-70-ms decrease in action potential duration. The data describe a novel mechanism by which hERG channel gating is modulated through physiologically and pathophysiologically relevant changes in N-glycosylation; reduced channel sialylation increases hERG channel activity during the action potential, thereby increasing the rate of action potential repolarization.

Glycosyl-Nucleolipids as New Bioinspired Amphiphiles

Directory of Open Access Journals (Sweden)

Philippe Barthélémy

2013-09-01

Full Text Available Four new Glycosyl-NucleoLipid (GNL analogs featuring either a single fluorocarbon or double hydrocarbon chains were synthesized in good yields from azido thymidine as starting material. Physicochemical studies (surface tension measurements, differential scanning calorimetry indicate that hydroxybutanamide-based GNLs feature endothermic phase transition temperatures like the previously reported double chain glycerol-based GNLs. The second generation of GNFs featuring a free nucleobase reported here presents a better surface activity (lower glim compared to the first generation of GNFs.

Deglycosylation systematically improves N-glycoprotein identification in liquid chromatography-tandem mass spectrometry proteomics for analysis of cell wall stress responses in Saccharomyces cerevisiae lacking Alg3p.

Science.gov (United States)

Bailey, Ulla-Maja; Schulz, Benjamin L

2013-04-01

Post-translational modification of proteins with glycosylation is of key importance in many biological systems in eukaryotes, influencing fundamental biological processes and regulating protein function. Changes in glycosylation are therefore of interest in understanding these processes and are also useful as clinical biomarkers of disease. The presence of glycosylation can also inhibit protease digestion and lower the quality and confidence of protein identification by mass spectrometry. While deglycosylation can improve the efficiency of subsequent protease digest and increase protein coverage, this step is often excluded from proteomic workflows. Here, we performed a systematic analysis that showed that deglycosylation with peptide-N-glycosidase F (PNGase F) prior to protease digestion with AspN or trypsin improved the quality of identification of the yeast cell wall proteome. The improvement in the confidence of identification of glycoproteins following PNGase F deglycosylation correlated with a higher density of glycosylation sites. Optimal identification across the proteome was achieved with PNGase F deglycosylation and complementary proteolysis with either AspN or trypsin. We used this combination of deglycosylation and complementary protease digest to identify changes in the yeast cell wall proteome caused by lack of the Alg3p protein, a key component of the biosynthetic pathway of protein N-glycosylation. The cell wall of yeast lacking Alg3p showed specifically increased levels of Cis3p, a protein important for cell wall integrity. Our results showed that deglycosylation prior to protease digestion improved the quality of proteomic analyses even if protein glycosylation is not of direct relevance to the study at hand. Copyright © 2013 Elsevier B.V. All rights reserved.

Site-specific O-glycosylation of members of the low-density lipoprotein receptor superfamily enhances ligand interactions.

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Wang, Shengjun; Mao, Yang; Narimatsu, Yoshiki; Ye, Zilu; Tian, Weihua; Goth, Christoffer K; Lira-Navarrete, Erandi; Pedersen, Nis B; Benito-Vicente, Asier; Martin, Cesar; Uribe, Kepa B; Hurtado-Guerrero, Ramon; Christoffersen, Christina; Seidah, Nabil G; Nielsen, Rikke; Christensen, Erik I; Hansen, Lars; Bennett, Eric P; Vakhrushev, Sergey Y; Schjoldager, Katrine T; Clausen, Henrik

2018-05-11

The low-density lipoprotein receptor (LDLR) and related receptors are important for the transport of diverse biomolecules across cell membranes and barriers. Their functions are especially relevant for cholesterol homeostasis and diseases, including neurodegenerative and kidney disorders. Members of the LDLR-related protein family share LDLR class A (LA) repeats providing binding properties for lipoproteins and other biomolecules. We previously demonstrated that short linker regions between these LA repeats contain conserved O -glycan sites. Moreover, we found that O -glycan modifications at these sites are selectively controlled by the GalNAc-transferase isoform, GalNAc-T11. However, the effects of GalNAc-T11-mediated O -glycosylation on LDLR and related receptor localization and function are unknown. Here, we characterized O -glycosylation of LDLR-related proteins and identified conserved O -glycosylation sites in the LA linker regions of VLDLR, LRP1, and LRP2 (Megalin) from both cell lines and rat organs. Using a panel of gene-edited isogenic cell line models, we demonstrate that GalNAc-T11-mediated LDLR and VLDLR O -glycosylation is not required for transport and cell-surface expression and stability of these receptors but markedly enhances LDL and VLDL binding and uptake. Direct ELISA-based binding assays with truncated LDLR constructs revealed that O -glycosylation increased affinity for LDL by ∼5-fold. The molecular basis for this observation is currently unknown, but these findings open up new avenues for exploring the roles of LDLR-related proteins in disease. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Electrostatics and N-glycan-mediated membrane tethering of SCUBE1 is critical for promoting bone morphogenetic protein signalling.

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Liao, Wei-Ju; Tsao, Ku-Chi; Yang, Ruey-Bing

2016-03-01

SCUBE1 (S1), a secreted and membrane-bound glycoprotein, has a modular protein structure composed of an N-terminal signal peptide sequence followed by nine epidermal growth factor (EGF)-like repeats, a spacer region and three cysteine-rich (CR) motifs with multiple potential N-linked glycosylation sites, and one CUB domain at the C-terminus. Soluble S1 is a biomarker of platelet activation but an active participant of thrombosis via its adhesive EGF-like repeats, whereas its membrane-associated form acts as a bone morphogenetic protein (BMP) co-receptor in promoting BMP signal activity. However, the mechanism responsible for the membrane tethering and the biological importance of N-glycosylation of S1 remain largely unknown. In the present study, molecular mapping analysis identified a polycationic segment (amino acids 501-550) in the spacer region required for its membrane tethering via electrostatic interactions possibly with the anionic heparan sulfate proteoglycans. Furthermore, deglycosylation by peptide N-glycosidase F treatment revealed that N-glycans within the CR motif are essential for membrane recruitment through lectin-mediated surface retention. Injection of mRNA encoding zebrafish wild-type but not N-glycan-deficient scube1 restores the expression of haematopoietic and erythroid markers (scl and gata1) in scube1-knockdown embryos. We describe novel mechanisms in targeting S1 to the plasma membrane and demonstrate that N-glycans are required for S1 functions during primitive haematopoiesis in zebrafish. © 2016 Authors; published by Portland Press Limited.

Differences in N-glycosylation of recombinant human coagulation factor VII derived from BHK, CHO, and HEK293 cells.

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Böhm, Ernst; Seyfried, Birgit K; Dockal, Michael; Graninger, Michael; Hasslacher, Meinhard; Neurath, Marianne; Konetschny, Christian; Matthiessen, Peter; Mitterer, Artur; Scheiflinger, Friedrich

2015-09-18

BACKGROUND & Recombinant factor VII (rFVII), the precursor molecule for recombinant activated FVII (rFVIIa), is, due to its need for complex post translational modifications, produced in mammalian cells. To evaluate the suitability of a human cell line in order to produce rFVII with post-translational modifications as close as possible to pdFVII, we compared the biochemical properties of rFVII synthesized in human embryonic kidney-derived (HEK)293 cells (HEK293rFVII) with those of rFVII expressed in Chinese hamster ovary (CHO, CHOrFVII) and baby hamster kidney (BHK, BHKrFVII) cells, and also with those of plasma derived FVII (pdFVII), using various analytical methods. rFVII was purified from selected production clones derived from BHK, CHO, and HEK293 cells after stable transfection, and rFVII isolates were analyzed for protein activity, impurities and post-translational modifications. RESULTS & The analytical results showed no apparent gross differences between the various FVII proteins, except in their N-linked glycosylation pattern. Most N-glycans found on rFVII produced in HEK293 cells were not detected on rFVII from CHO and BHK cells, or, somewhat unexpectedly, on pdFVII; all other protein features were similar. HEK293rFVII glycans were mainly characterized by a higher structural variety and a lower degree of terminal sialylation, and a high amount of terminal N-acetyl galactosamines (GalNAc). All HEK293rFVII oligosaccharides contained one or more fucoses (Fuc), as well as hybrid and high mannose (Man) structures. From all rFVII isolates investigated, CHOrFVII contained the highest degree of sialylation and no terminal GalNAc, and CHO cells were therefore assumed to be the best option for the production of rFVII.

CCDC115 Deficiency Causes a Disorder of Golgi Homeostasis with Abnormal Protein Glycosylation.

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Jansen, Jos C; Cirak, Sebahattin; van Scherpenzeel, Monique; Timal, Sharita; Reunert, Janine; Rust, Stephan; Pérez, Belén; Vicogne, Dorothée; Krawitz, Peter; Wada, Yoshinao; Ashikov, Angel; Pérez-Cerdá, Celia; Medrano, Celia; Arnoldy, Andrea; Hoischen, Alexander; Huijben, Karin; Steenbergen, Gerry; Quelhas, Dulce; Diogo, Luisa; Rymen, Daisy; Jaeken, Jaak; Guffon, Nathalie; Cheillan, David; van den Heuvel, Lambertus P; Maeda, Yusuke; Kaiser, Olaf; Schara, Ulrike; Gerner, Patrick; van den Boogert, Marjolein A W; Holleboom, Adriaan G; Nassogne, Marie-Cécile; Sokal, Etienne; Salomon, Jody; van den Bogaart, Geert; Drenth, Joost P H; Huynen, Martijn A; Veltman, Joris A; Wevers, Ron A; Morava, Eva; Matthijs, Gert; Foulquier, François; Marquardt, Thorsten; Lefeber, Dirk J

2016-02-04

Disorders of Golgi homeostasis form an emerging group of genetic defects. The highly heterogeneous clinical spectrum is not explained by our current understanding of the underlying cell-biological processes in the Golgi. Therefore, uncovering genetic defects and annotating gene function are challenging. Exome sequencing in a family with three siblings affected by abnormal Golgi glycosylation revealed a homozygous missense mutation, c.92T>C (p.Leu31Ser), in coiled-coil domain containing 115 (CCDC115), the function of which is unknown. The same mutation was identified in three unrelated families, and in one family it was compound heterozygous in combination with a heterozygous deletion of CCDC115. An additional homozygous missense mutation, c.31G>T (p.Asp11Tyr), was found in a family with two affected siblings. All individuals displayed a storage-disease-like phenotype involving hepatosplenomegaly, which regressed with age, highly elevated bone-derived alkaline phosphatase, elevated aminotransferases, and elevated cholesterol, in combination with abnormal copper metabolism and neurological symptoms. Two individuals died of liver failure, and one individual was successfully treated by liver transplantation. Abnormal N- and mucin type O-glycosylation was found on serum proteins, and reduced metabolic labeling of sialic acids was found in fibroblasts, which was restored after complementation with wild-type CCDC115. PSI-BLAST homology detection revealed reciprocal homology with Vma22p, the yeast V-ATPase assembly factor located in the endoplasmic reticulum (ER). Human CCDC115 mainly localized to the ERGIC and to COPI vesicles, but not to the ER. These data, in combination with the phenotypic spectrum, which is distinct from that associated with defects in V-ATPase core subunits, suggest a more general role for CCDC115 in Golgi trafficking. Our study reveals CCDC115 deficiency as a disorder of Golgi homeostasis that can be readily identified via screening for abnormal

The glycosylation and characterization of the candidate Gc macrophage activating factor.

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Ravnsborg, Tina; Olsen, Dorthe T; Thysen, Anna Hammerich; Christiansen, Maja; Houen, Gunnar; Højrup, Peter

2010-04-01

The vitamin D binding protein, Gc globulin, has in recent years received some attention for its role as precursor for the extremely potent macrophage activating factor (GcMAF). An O-linked trisaccharide has been allocated to the threonine residue at position 420 in two of the three most common isoforms of Gc globulin (Gc1s and Gc1f). A substitution for a lysine residue at position 420 in Gc2 prevents this isoform from being glycosylated at that position. It has been suggested that Gc globulin subjected sequentially to sialidase and galactosidase treatment generates GcMAF in the form of Gc globulin with only a single GalNAc attached to T420. In this study we confirm the location of a linear trisaccharide on T420. Furthermore, we provide the first structural evidence of the generation of the proposed GcMAF by use of glycosidase treatment and mass spectrometry. Additionally the generated GcMAF candidate was tested for its effect on cytokine release from macrophages in human whole blood. Copyright 2010 Elsevier B.V. All rights reserved.

Glycosylation-Based Serum Biomarkers for Cancer Diagnostics and Prognostics.

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Kirwan, Alan; Utratna, Marta; O'Dwyer, Michael E; Joshi, Lokesh; Kilcoyne, Michelle

2015-01-01

Cancer is the second most common cause of death in developed countries with approximately 14 million newly diagnosed individuals and over 6 million cancer-related deaths in 2012. Many cancers are discovered at a more advanced stage but better survival rates are correlated with earlier detection. Current clinically approved cancer biomarkers are most effective when applied to patients with widespread cancer. Single biomarkers with satisfactory sensitivity and specificity have not been identified for the most common cancers and some biomarkers are ineffective for the detection of early stage cancers. Thus, novel biomarkers with better diagnostic and prognostic performance are required. Aberrant protein glycosylation is well known hallmark of cancer and represents a promising source of potential biomarkers. Glycoproteins enter circulation from tissues or blood cells through active secretion or leakage and patient serum is an attractive option as a source for biomarkers from a clinical and diagnostic perspective. A plethora of technical approaches have been developed to address the challenges of glycosylation structure detection and determination. This review summarises currently utilised glycoprotein biomarkers and novel glycosylation-based biomarkers from the serum glycoproteome under investigation as cancer diagnostics and for monitoring and prognostics and includes details of recent high throughput and other emerging glycoanalytical techniques.

Understanding Alzheimer's disease by global quantification of protein phosphorylation and sialylated N-linked glycosylation profiles

DEFF Research Database (Denmark)

Lassen, Pernille S.; Thygesen, Camilla; Larsen, Martin R.

2017-01-01

elucidated them in neurodegenerative diseases such as Alzheimer's disease. Here, we comprehensively review Alzheimer's pathology in relation to protein phosphorylation and glycosylation on synaptic plasticity from neuroproteomics data. Moreover, we highlight several mass spectrometry-based sample processing...

Optimized expression of prolyl aminopeptidase in Pichia pastoris and its characteristics after glycosylation.

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Yang, Hongyu; Zhu, Qiang; Zhou, Nandi; Tian, Yaping

2016-11-01

Prolyl aminopeptidases are specific exopeptidases that catalyze the hydrolysis of the N-terminus proline residue of peptides and proteins. In the present study, the prolyl aminopeptidase gene (pap) from Aspergillus oryzae JN-412 was optimized through the codon usage of Pichia pastoris. Both the native and optimized pap genes were inserted into the expression vector pPIC9 K and were successfully expressed in P. pastoris. Additionally, the activity of the intracellular enzyme expressed by the recombinant optimized pap gene reached 61.26 U mL(-1), an activity that is 2.1-fold higher than that of the native gene. The recombinant enzyme was purified by one-step elution through Ni-affinity chromatography. The optimal temperature and pH of the purified PAP were 60 °C and 7.5, respectively. Additionally, the recombinant PAP was recovered at a yield greater than 65 % at an extremely broad range of pH values from 6 to 10 after treatment at 50 °C for 6 h. The molecular weight of the recombinant PAP decreased from 50 kDa to 48 kDa after treatment with a deglycosylation enzyme, indicating that the recombinant PAP was completely glycosylated. The glycosylated PAP exhibited high thermo-stability. Half of the activity remained after incubation at 50 °C for 50 h, whereas the remaining activity of PAP expressed in E. coli was only 10 % after incubation at 50 °C for 1 h. PAP could be activated by the appropriate salt concentration and exhibited salt tolerance against NaCl at a concentration up to 5 mol L(-1).

Composição química e atividades biológicas das folhas de Cynara scolymus L. (alcachofra cultivada no Brasil Chemical composition and biological activities of the leaves of Cynara scolymus L. (artichoke cultivated in Brazil

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Vânia Floriani Noldin

2003-05-01

Full Text Available The present paper describes the chemical composition and biological activities of artichoke cultivated in Brazil. Our studies demonstrated that glycosyl flavonoids (cynaroside and scolymoside, are the major constituents, along with cynaropicrin, a sesquiterpene lactone, and the triterpene lupeol. Cynarin, which is the main compound described for artichoke, was detected in very low concentration. Hexanic fraction exhibited considerable cytotoxicity and diuretic activities.

Structural analysis of PseH, the Campylobacter jejuni N-acetyltransferase involved in bacterial O-linked glycosylation

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Song, Wan Seok; Nam, Mi Sun; Namgung, Byeol [Department of Systems Immunology, College of Biomedical Science, Kangwon National University, Chuncheon 200-701 (Korea, Republic of); Yoon, Sung-il, E-mail: sungil@kangwon.ac.kr [Department of Systems Immunology, College of Biomedical Science, Kangwon National University, Chuncheon 200-701 (Korea, Republic of); Institute of Bioscience and Biotechnology, Kangwon National University, Chuncheon 200-701 (Korea, Republic of)

2015-03-20

Campylobacter jejuni is a bacterium that uses flagella for motility and causes worldwide acute gastroenteritis in humans. The C. jejuni N-acetyltransferase PseH (cjPseH) is responsible for the third step in flagellin O-linked glycosylation and plays a key role in flagellar formation and motility. cjPseH transfers an acetyl group from an acetyl donor, acetyl coenzyme A (AcCoA), to the amino group of UDP-4-amino-4,6-dideoxy-N-acetyl-β-L-altrosamine to produce UDP-2,4-diacetamido-2,4,6-trideoxy-β-L-altropyranose. To elucidate the catalytic mechanism of cjPseH, crystal structures of cjPseH alone and in complex with AcCoA were determined at 1.95 Å resolution. cjPseH folds into a single-domain structure of a central β-sheet decorated by four α-helices with two continuously connected grooves. A deep groove (groove-A) accommodates the AcCoA molecule. Interestingly, the acetyl end of AcCoA points toward an open space in a neighboring shallow groove (groove-S), which is occupied by extra electron density that potentially serves as a pseudosubstrate, suggesting that the groove-S may provide a substrate-binding site. Structure-based comparative analysis suggests that cjPseH utilizes a unique catalytic mechanism of acetylation that has not been observed in other glycosylation-associated acetyltransferases. Thus, our studies on cjPseH will provide valuable information for the design of new antibiotics to treat C. jejuni-induced gastroenteritis. - Highlights: • cjPseH adopts a single-domain structure of a central β-sheet decorated by α-helices. • cjPseH features two continuously connected grooves on the protein surface. • Acetyl coenzyme A (AcCoA) binds into a deep groove of cjPseH in an ‘L’ shape. • The acetyl end of AcCoA points to a wide groove, a potential substrate-binding site.

Glycosylated yellow laccases of the basidiomycete Stropharia aeruginosa.

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Daroch, Maurycy; Houghton, Catharine A; Moore, Jonathan K; Wilkinson, Mark C; Carnell, Andrew J; Bates, Andrew D; Iwanejko, Lesley A

2014-05-10

Here we describe the identification, purification and characterisation of glycosylated yellow laccase proteins from the basidiomycete fungus Stropharia aeruginosa. Biochemical characterisation of two yellow laccases, Yel1p and Yel3p, show that they are both secreted, monomeric, N-glycosylated proteins of molecular weight around 55kDa with substrate specificities typical of laccases, but lacking the absorption band at 612nm typical of the blue laccase proteins. Low coverage, high throughput 454 transcriptome sequencing in combination with inverse-PCR was used to identify cDNA sequences. One of the cDNA sequences has been assigned to the Yel1p protein on the basis of identity between the translated protein sequence and the peptide data from the purified protein, and the full length gene sequence has been obtained. Biochemical properties, substrate specificities and protein sequence data have been used to discuss the unusual spectroscopic properties of S. aeruginosa proteins in the context of recent theories about the differences between yellow and blue laccases. Copyright © 2014 Elsevier Inc. All rights reserved.

Chapter Three -- Glycosylation of Cellulases: Engineering Better Enzymes for Biofuels

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Greene, Eric R. [Univ. of Colorado, Boulder, CO (United States). Dept. of Chemistry and Biochemistry and BioFrontiers Inst.; Himmel, Michael E. [National Renewable Energy Lab. (NREL), Golden, CO (United States). Biosciences Center; Beckham, Gregg T. [National Renewable Energy Lab. (NREL), Golden, CO (United States). National Bioenergy Center; Tan, Zhongping [Univ. of Colorado, Boulder, CO (United States). Dept. of Chemistry and Biochemistry and BioFrontiers Inst.

2015-10-24

Methods for the manipulation of glycan structures have been recently reported that employ genetic tuning of glycan-active enzymes expressed from homogeneous and heterologous fungal hosts. Taken together, these studies have enabled new strategies for the exploitation of protein glycosylation for the production of enhanced cellulases for biofuel production.

Identification of the Mycobacterium marinum Apa antigen O-mannosylation sites reveals important glycosylation variability with the M. tuberculosis Apa homologue.

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Coddeville, Bernadette; Wu, Sz-Wei; Fabre, Emeline; Brassart, Colette; Rombouts, Yoann; Burguière, Adeline; Kremer, Laurent; Khoo, Kay-Hooi; Elass-Rochard, Elisabeth; Guérardel, Yann

2012-10-22

The 45/47 kDa Apa, an immuno-dominant antigen secreted by Mycobacterium tuberculosis is O-mannosylated at multiple sites. Glycosylation of Apa plays a key role in colonization and invasion of the host cells by M. tuberculosis through interactions of Apa with the host immune system C-type lectins. Mycobacterium marinum (M.ma) a fish pathogen, phylogenetically close to M. tuberculosis, induces a granulomatous response with features similar to those described for M. tuberculosis in human. Although M.ma possesses an Apa homologue, its glycosylation status is unknown, and whether this represents a crucial element in the pathophysiology induced by M.ma remains to be addressed. To this aim, we have identified two concanavalin A-reactive 45/47 kDa proteins from M.ma, which have been further purified by a two-step anion exchange chromatography process. Advanced liquid chromatography-nanoESI mass spectrometry-based proteomic analyses of peptides, derived from either tryptic digestion alone or in combination with the Asp-N endoproteinase, established that M.ma Apa possesses up to seven distinct O-mannosylated sites with mainly single mannose substitutions, which can be further extended at the Ser/Thr/Pro rich region near the N-terminus. This opens the way to further studies focussing on the involvement and biological functions of Apa O-mannosylation using the M.ma/zebrafish model. Copyright © 2012 Elsevier B.V. All rights reserved.

Effects of macro nutrient concentration on biological N2 fixation by Azotobacter vinelandii ATCC 12837

International Nuclear Information System (INIS)

Liew Pauline Woan Ying; Nazalan Najimudin; Jong Bor Chyan; Latiffah Noordin; Khairuddin Abdul Rahim; Amir Hamzah Ahmad Ghazali

2010-01-01

The dynamic changes of biological N 2 fixation by Azotobacter vinelandii ATCC 12837 under the influence of various macro nutrients, specifically phosphorus (P) and potassium (K), was investigated. In this attempt, Oryza sativa L. var. MR 219 was used as the model plant. Results obtained showed changes in the biological N 2 fixation activities with different macro nutrient(s) manipulations. The research activity enables optimisation of macro nutrients concentration for optimal/ enhanced biological N 2 fixation by A. vinelandii ATCC 12837. (author)

Rev1 Recruits Ung to Switch Regions and Enhances dU Glycosylation for Immunoglobulin Class Switch DNA Recombination

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Hong Zan

2012-11-01

Full Text Available By diversifying the biological effector functions of antibodies, class switch DNA recombination (CSR plays a critical role in the maturation of the immune response. It is initiated by activation-induced cytidine deaminase (AID-mediated deoxycytosine deamination, yielding deoxyuridine (dU, and dU glycosylation by uracil DNA glycosylase (Ung in antibody switch (S region DNA. Here we showed that the translesion DNA synthesis polymerase Rev1 directly interacted with Ung and targeted in an AID-dependent and Ung-independent fashion the S regions undergoing CSR. Rev1−/− Ung+/+ B cells reduced Ung recruitment to S regions, DNA-dU glycosylation, and CSR. Together with an S region spectrum of mutations similar to that of Rev1+/+ Ung−/− B cells, this suggests that Rev1 operates in the same pathway as Ung, as emphasized by further decreased CSR in Rev1−/− Msh2−/− B cells. Rescue of CSR in Rev1−/− B cells by a catalytically inactive Rev1 mutant shows that the important role of Rev1 in CSR is mediated by Rev1’s scaffolding function, not its enzymatic function.

Characterization of Thermo- and Detergent Stable Antigenic Glycosylated Cysteine Protease of Euphorbia nivulia Buch.-Ham. and Evaluation of Its Ecofriendly Applications

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Shamkant B. Badgujar

2013-01-01

Full Text Available An antigenic glycosylated cysteine protease has been purified from the latex of Euphorbia nivulia Buch.-Ham. It exhibits remarkable protease activity in the presence of metal ions, oxidizing agents, organic solvents, and detergents. This enzyme showed potential role in leather processing industry due to its dehairing activity for animal hide without hydrolyzing fibrous proteins, producing, by this way, a better quality product. The enzyme can also be used for silver recovering from X-ray plates. In addition, the stability (temperature and surfactants and hydrolysis of blood stain data also revealed its application in detergent industries. Agriculturally, this protease finds application in biocontrol process against the infectious management of root knot nematode, Meloidogyne incognita. Biologically, it shows noticeable wound healing, haemostatic and antibacterial activity.

N-glycan engineering of a plant-produced anti-CD20-hIL-2 immunocytokine significantly enhances its effector functions.

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Marusic, Carla; Pioli, Claudio; Stelter, Szymon; Novelli, Flavia; Lonoce, Chiara; Morrocchi, Elena; Benvenuto, Eugenio; Salzano, Anna Maria; Scaloni, Andrea; Donini, Marcello

2018-03-01

Anti-CD20 recombinant antibodies are among the most promising therapeutics for the treatment of B-cell malignancies such as non-Hodgkin lymphomas. We recently demonstrated that an immunocytokine (2B8-Fc-hIL2), obtained by fusing an anti-CD20 scFv-Fc antibody derived from C2B8 mAb (rituximab) to the human interleukin 2 (hIL-2), can be efficiently produced in Nicotiana benthamiana plants. The purified immunocytokine (IC) bearing a typical plant protein N-glycosylation profile showed a CD20 binding activity comparable to that of rituximab and was efficient in eliciting antibody-dependent cell-mediated cytotoxicity (ADCC) of human PBMC against Daudi cells, indicating its fuctional integrity. In this work, the immunocytokine devoid of the typical xylose/fucose N-glycosylation plant signature (IC-ΔXF) and the corresponding scFv-Fc-ΔXF antibody not fused to the cytokine, were obtained in a glyco-engineered ΔXylT/FucT N. benthamiana line. Purification yields from agroinfiltrated plants amounted to 20-35 mg/kg of leaf fresh weight. When assayed for interaction with FcγRI and FcγRIIIa, IC-ΔXF exhibited significantly enhanced binding affinities if compared to the counterpart bearing the typical plant protein N-glycosylation profile (IC) and to rituximab. The glyco-engineered recombinant molecules also exhibited a strongly improved ADCC and complement-dependent cytotoxicity (CDC). Notably, our results demonstrate a reduced C1q binding of xylose/fucose carrying IC and scFv-Fc compared to versions that lack these sugar moieties. These results demonstrate that specific N-glycosylation alterations in recombinant products can dramatically affect the effector functions of the immunocytokine, resulting in an overall improvement of the biological functions and consequently of the therapeutic potential. © 2017 Wiley Periodicals, Inc.

N-linked glycosylation of SV2 is required for binding and uptake of botulinum neurotoxin A

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Yao, Guorui; Zhang, Sicai; Mahrhold, Stefan; Lam, Kwok-ho; Stern, Daniel; Bagramyan, Karine; Perry, Kay; Kalkum, Markus; Rummel, Andreas; Dong, Min; Jin, Rongsheng

2016-01-01

Botulinum neurotoxin serotype A1 (BoNT/A1) is one of the most dangerous potential bioterrorism agents, and exerts its action by invading motoneurons. It is also a licensed drug widely used for medical and cosmetic applications. Here we report a 2.0 Å resolution crystal structure of BoNT/A1 receptor-binding domain in complex with its neuronal receptor, the glycosylated human SV2C. We find that the neuronal tropism of BoNT/A1 requires recognition of both the peptide moiety and an N-linked glycan on SV2. This N-glycan—conserved in all SV2 isoforms across vertebrates—is essential for BoNT/A1 binding to neurons and its potent neurotoxicity. The glycan-binding interface on SV2 is targeted by a human BoNT/A1-neutralizing antibody currently licensed as an anti-botulism drug. Our studies reveal a new paradigm of host-pathogen interactions, in which pathogens exploit conserved host post-translational modifications to achieve highly specific receptor binding while also tolerating genetic changes across multiple isoforms of receptors. PMID:27294781

The role of glycosylation in breast cancer metastasis and cancer control

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Alexandra eKölbl

2015-10-01

Full Text Available AbstractGlycosylation and its correlation to the formation of remote metastasis in breast cancer had been an important scientific topic in the last 25 years. With the development of new analytical techniques new insights were gained on the mechanisms underlying metastasis formation and the role of aberrant glycosylation within. Mucin-1 and Galectin were recognized as key players in glycosylation. Interestingly, aberrant carbohydrate structures seem to support the development of brain metastasis in breast cancer patients, as changes in glycosylation structures facilitate an overcoming of blood-brain barrier. Changes in the gene expression of glycosyltransferases are the leading cause for a modification of carbohydrate chains, so that also altered gene expression plays a role for glycosylation. In consequence, glycosylation and changes within can be useful for cancer diagnosis, determination of tumour stage and prognosis, but can as well be targets for therapeutic strategies. Thus, further research on this topic would worth wile for cancer combating.

Mining the Virgin Land of Neurotoxicology: A Novel Paradigm of Neurotoxic Peptides Action on Glycosylated Voltage-Gated Sodium Channels

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Zhirui Liu

2012-01-01

Full Text Available Voltage-gated sodium channels (VGSCs are important membrane protein carrying on the molecular basis for action potentials (AP in neuronal firings. Even though the structure-function studies were the most pursued spots, the posttranslation modification processes, such as glycosylation, phosphorylation, and alternative splicing associating with channel functions captured less eyesights. The accumulative research suggested an interaction between the sialic acids chains and ion-permeable pores, giving rise to subtle but significant impacts on channel gating. Sodium channel-specific neurotoxic toxins, a family of long-chain polypeptides originated from venomous animals, are found to potentially share the binding sites adjacent to glycosylated region on VGSCs. Thus, an interaction between toxin and glycosylated VGSC might hopefully join the campaign to approach the role of glycosylation in modulating VGSCs-involved neuronal network activity. This paper will cover the state-of-the-art advances of researches on glycosylation-mediated VGSCs function and the possible underlying mechanisms of interactions between toxin and glycosylated VGSCs, which may therefore, fulfill the knowledge in identifying the pharmacological targets and therapeutic values of VGSCs.

Identification of a general O-linked protein glycosylation system in Acinetobacter baumannii and its role in virulence and biofilm formation.

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Jeremy A Iwashkiw

Full Text Available Acinetobacter baumannii is an emerging cause of nosocomial infections. The isolation of strains resistant to multiple antibiotics is increasing at alarming rates. Although A. baumannii is considered as one of the more threatening "superbugs" for our healthcare system, little is known about the factors contributing to its pathogenesis. In this work we show that A. baumannii ATCC 17978 possesses an O-glycosylation system responsible for the glycosylation of multiple proteins. 2D-DIGE and mass spectrometry methods identified seven A. baumannii glycoproteins, of yet unknown function. The glycan structure was determined using a combination of MS and NMR techniques and consists of a branched pentasaccharide containing N-acetylgalactosamine, glucose, galactose, N-acetylglucosamine, and a derivative of glucuronic acid. A glycosylation deficient strain was generated by homologous recombination. This strain did not show any growth defects, but exhibited a severely diminished capacity to generate biofilms. Disruption of the glycosylation machinery also resulted in reduced virulence in two infection models, the amoebae Dictyostelium discoideum and the larvae of the insect Galleria mellonella, and reduced in vivo fitness in a mouse model of peritoneal sepsis. Despite A. baumannii genome plasticity, the O-glycosylation machinery appears to be present in all clinical isolates tested as well as in all of the genomes sequenced. This suggests the existence of a strong evolutionary pressure to retain this system. These results together indicate that O-glycosylation in A. baumannii is required for full virulence and therefore represents a novel target for the development of new antibiotics.

Identification of a General O-linked Protein Glycosylation System in Acinetobacter baumannii and Its Role in Virulence and Biofilm Formation

Science.gov (United States)

Iwashkiw, Jeremy A.; Seper, Andrea; Weber, Brent S.; Scott, Nichollas E.; Vinogradov, Evgeny; Stratilo, Chad; Reiz, Bela; Cordwell, Stuart J.; Whittal, Randy; Schild, Stefan; Feldman, Mario F.

2012-01-01

Acinetobacter baumannii is an emerging cause of nosocomial infections. The isolation of strains resistant to multiple antibiotics is increasing at alarming rates. Although A. baumannii is considered as one of the more threatening “superbugs” for our healthcare system, little is known about the factors contributing to its pathogenesis. In this work we show that A. baumannii ATCC 17978 possesses an O-glycosylation system responsible for the glycosylation of multiple proteins. 2D-DIGE and mass spectrometry methods identified seven A. baumannii glycoproteins, of yet unknown function. The glycan structure was determined using a combination of MS and NMR techniques and consists of a branched pentasaccharide containing N-acetylgalactosamine, glucose, galactose, N-acetylglucosamine, and a derivative of glucuronic acid. A glycosylation deficient strain was generated by homologous recombination. This strain did not show any growth defects, but exhibited a severely diminished capacity to generate biofilms. Disruption of the glycosylation machinery also resulted in reduced virulence in two infection models, the amoebae Dictyostelium discoideum and the larvae of the insect Galleria mellonella, and reduced in vivo fitness in a mouse model of peritoneal sepsis. Despite A. baumannii genome plasticity, the O-glycosylation machinery appears to be present in all clinical isolates tested as well as in all of the genomes sequenced. This suggests the existence of a strong evolutionary pressure to retain this system. These results together indicate that O-glycosylation in A. baumannii is required for full virulence and therefore represents a novel target for the development of new antibiotics. PMID:22685409

Does grazing of cover crops impact biologically active soil C and N fractions under inversion and no tillage management

Science.gov (United States)

Cover crops are a key component of conservation cropping systems. They can also be a key component of integrated crop-livestock systems by offering high-quality forage during short periods between cash crops. The impact of cattle grazing on biologically active soil C and N fractions has not receiv...

Inhibitory potential of pure isoflavonoids, red clover, and alfalfa extracts on hemoglobin glycosylation

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Mohsen Hosseini

2015-03-01

Full Text Available BACKGROUND: Non-enzymatic glycosylation of hemoglobin is complications of diabetes. Antioxidant system imbalance can result in the emergence of free radicals’ destructive effects in the long-term. Red clover (Trifolium pratense L. and alfalfa (Medicago sativa L. contain isoflavonoids and have antioxidant activity. This experimental study evaluated the inhibitory activity of pure isoflavonoids (daidzein and genistein, red clover and alfalfa extracts on hemoglobin glycosylation. METHODS: This study was performed in Iran. Stock solution of hydroalcoholic extracts of red clover and alfalfa in concentrations of 1 and 10 g/100 ml and stock solution of daidzein and genistein in concentrations of 250 ng, 500 ng, 25 µg and 250 µg/100 ml were prepared as case groups. Control group was without hydroalcoholic extracts of plants and pure isoflavonoids. All experiments were performed in triplicate. Hemoglobin was prepared and antioxidant activities were investigated to estimate degree of nonenzymatic hemoglobin glycosylation. RESULTS: There was no significantly difference between used extracts (extract of red clover and alfalfa and control of the hemoglobin glycosylation but using daidzein (P = 0.046, 0.029 and 0.021, respectively and genistein (P = 0.034, 0.036 and 0.028 significantly inhibited (P < 0.050 this reaction in 25 µg/100 ml, 250 and 500 ng/100 ml concentrations when compared to control. in 25 µg/100 ml, 250 ng and 500 ng/100 ml concentrations percentage of inhibition were 32, 80 and 74.5% respectively with used of daidzein and were 21, 83 and 76% respectively with consumption of genistein. CONCLUSION: According to decrease of glycation of hemoglobin with isoflavonoids, two used plant in this study containing isoflavonoid may be useful on diabetes.   

Ultra-Fast Glyco-Coating of Non-Biological Surfaces

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Eleanor Williams

2016-01-01

Full Text Available The ability to glycosylate surfaces has medical and diagnostic applications, but there is no technology currently recognized as being able to coat any surface without the need for prior chemical modification of the surface. Recently, a family of constructs called function-spacer-lipids (FSL has been used to glycosylate cells. Because it is known that lipid-based material can adsorb onto surfaces, we explored the potential and performance of cell-labelling FSL constructs to “glycosylate” non-biological surfaces. Using blood group A antigen as an indicator, the performance of a several variations of FSL constructs to modify a large variety of non-biological surfaces was evaluated. It was found the FSL constructs when optimised could in a few seconds glycosylate almost any non-biological surface including metals, glass, plastics, rubbers and other polymers. Although the FSL glycan coating was non-covalent, and therefore temporary, it was sufficiently robust with appropriate selection of spacer and surface that it could capture anti-glycan antibodies, immobilize cells (via antibody, and withstand incubation in serum and extensive buffer washing, making it suitable for diagnostic and research applications.

Antimitotic antitumor agents: synthesis, structure-activity relationships, and biological characterization of N-aryl-N'-(2-chloroethyl)ureas as new selective alkylating agents.

Science.gov (United States)

Mounetou, E; Legault, J; Lacroix, J; C-Gaudreault, R

2001-03-01

A series of N-aryl-N'-(2-chloroethyl)ureas (CEUs) and derivatives were synthesized and evaluated for antiproliferative activity against a wide panel of tumor cell lines. Systematic structure--activity relationship (SAR) studies indicated that: (i) a branched alkyl chain or a halogen at the 4-position of the phenyl ring or a fluorenyl/indanyl group, (ii) an exocyclic urea function, and (iii) a N'-2-chloroethyl moiety were required to ensure significant cytotoxicity. Biological experiments, such as immunofluorescence microscopy, confirmed that these promising compounds alter the cytoskeleton by inducing microtubule depolymerization via selective alkylation of beta-tubulin. Subsequent evaluations demonstrated that potent CEUs were weak alkylators, were non-DNA-damaging agents, and did not interact with the thiol function of either glutathione or glutathione reductase. Therefore, CEUs are part of a new class of antimitotic agents. Finally, among the series of CEUs evaluated, compounds 12, 15, 16, and 27 were selected for further in vivo trials.

Surface glycosylation profiles of urine extracellular vesicles.

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Jared Q Gerlach

Full Text Available Urinary extracellular vesicles (uEVs are released by cells throughout the nephron and contain biomolecules from their cells of origin. Although uEV-associated proteins and RNA have been studied in detail, little information exists regarding uEV glycosylation characteristics. Surface glycosylation profiling by flow cytometry and lectin microarray was applied to uEVs enriched from urine of healthy adults by ultracentrifugation and centrifugal filtration. The carbohydrate specificity of lectin microarray profiles was confirmed by competitive sugar inhibition and carbohydrate-specific enzyme hydrolysis. Glycosylation profiles of uEVs and purified Tamm Horsfall protein were compared. In both flow cytometry and lectin microarray assays, uEVs demonstrated surface binding, at low to moderate intensities, of a broad range of lectins whether prepared by ultracentrifugation or centrifugal filtration. In general, ultracentrifugation-prepared uEVs demonstrated higher lectin binding intensities than centrifugal filtration-prepared uEVs consistent with lesser amounts of co-purified non-vesicular proteins. The surface glycosylation profiles of uEVs showed little inter-individual variation and were distinct from those of Tamm Horsfall protein, which bound a limited number of lectins. In a pilot study, lectin microarray was used to compare uEVs from individuals with autosomal dominant polycystic kidney disease to those of age-matched controls. The lectin microarray profiles of polycystic kidney disease and healthy uEVs showed differences in binding intensity of 6/43 lectins. Our results reveal a complex surface glycosylation profile of uEVs that is accessible to lectin-based analysis following multiple uEV enrichment techniques, is distinct from co-purified Tamm Horsfall protein and may demonstrate disease-specific modifications.

Synthesis, crystal structure and biological activity of n-(5-(o-tolyl)-1, 3, 4-thiadiazol-2-yl)cyclopropanecarboxamide

International Nuclear Information System (INIS)

Tong, J.Y.; Sun, N.B.; Wu, H.K.

2013-01-01

A new 1, 3, 4-thiadiazole compound, N-(5-(o-tolyl)-1,3,4-thiadiazol-2-yl) cyclopropanecarboxamide, was synthesized and its structure was confirmed by 1H NMR, MS and HRMS. The single crystal structure of the title compound was determined by X-ray diffraction. The preliminary biological test showed that the synthesized compound has moderate herbicidal activity against Brassica campestris and fungicidal activities against Sclerotinia sclerotiorum(Lib.) de Bary, Rhizoctonia solanii, Fusarium oxysporum, Corynespora cassiicola, and Botrytis cinerea. (author)

Protein glycosylation in cancers and its potential therapeutic applications in neuroblastoma

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Wan-Ling Ho

2016-09-01

Full Text Available Abstract Glycosylation is the most complex post-translational modification of proteins. Altered glycans on the tumor- and host-cell surface and in the tumor microenvironment have been identified to mediate critical events in cancer pathogenesis and progression. Tumor-associated glycan changes comprise increased branching of N-glycans, higher density of O-glycans, generation of truncated versions of normal counterparts, and generation of unusual forms of terminal structures arising from sialylation and fucosylation. The functional role of tumor-associated glycans (Tn, sTn, T, and sLea/x is dependent on the interaction with lectins. Lectins are expressed on the surface of immune cells and endothelial cells or exist as extracellular matrix proteins and soluble adhesion molecules. Expression of tumor-associated glycans is involved in the dysregulation of glycogenes, which mainly comprise glycosyltransferases and glycosidases. Furthermore, genetic and epigenetic mechanisms on many glycogenes are associated with malignant transformation. With better understanding of all aspects of cancer-cell glycomics, many tumor-associated glycans have been utilized for diagnostic, prognostic, and therapeutic purposes. Glycan-based therapeutics has been applied to cancers from breast, lung, gastrointestinal system, melanomas, and lymphomas but rarely to neuroblastomas (NBs. The success of anti-disialoganglioside (GD2, a glycolipid antigen antibodies sheds light on glycan-based therapies for NB and also suggests the possibility of protein glycosylation-based therapies for NB. This review summarizes our understanding of cancer glycobiology with a focus of how protein glycosylation and associated glycosyltransferases affect cellular behaviors and treatment outcome of various cancers, especially NB. Finally, we highlight potential applications of glycosylation in drug and cancer vaccine development for NB.

Quantifying risk of penile prosthesis infection with elevated glycosylated hemoglobin.

Science.gov (United States)

Wilson, S K; Carson, C C; Cleves, M A; Delk, J R

1998-05-01

Elevation of glycosylated hemoglobin above levels of 11.5 mg.% has been considered a contraindication to penile prosthesis implantation in diabetic patients. We determine the predictive value of glycosylated hemoglobin A1C in penile prosthesis infections in diabetic and nondiabetic patients to confirm or deny this prevalent opinion. We conducted a 2-year prospective study of 389 patients, including 114 diabetics, who underwent 3-piece penile prosthesis implantation. All patients had similar preoperative preparation without regard to diabetic status, control or glycosylated hemoglobin A1C level. Risk of infection was statistically analyzed for diabetics versus nondiabetics, glycosylated hemoglobin A1C values above and below 11.5 mg.%, insulin dependent versus oral medication diabetics, and fasting blood sugars above and below 180 mg.%. Prosthesis infections developed in 10 diabetics (8.7%) and 11 nondiabetics (4.0%). No increased infection rate was observed in diabetics with high fasting sugars or diabetics on insulin. There was no statistically significant increased infection risk with increased levels of glycosylated hemoglobin A1C among all patients or among only the diabetics. In fact, there was no meaningful difference in the median or mean level of glycosylated hemoglobin A1C in the infected and noninfected patients regardless of diabetes. Use of glycosylated hemoglobin A1C values to identify and exclude surgical candidates with increased risk of infections is not proved by this study. Elevation of fasting sugar or insulin dependence also does not increase risk of infection in diabetics undergoing prosthesis implantation.

Method Development in the Regioselective Glycosylation of Unprotected Carbohydrates

DEFF Research Database (Denmark)

Niedbal, Dominika Alina

and the glycosylations were promoted by tetrabutylammonium bromide. The couplings were completely selective and gave rise to a number of 1,6-linked disaccharides with 1,2- cis-linked orientation. Project 2: Boron-mediated glycosylation of unprotected carbohydrates Boron-mediated regioselective Koenigs...

Elucidating heterogeneity of IgA1 hinge-region O-glycosylation by use of MALDI-TOF/TOF mass spectrometry: role of cysteine alkylation during sample processing.

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Franc, Vojtěch; Řehulka, Pavel; Raus, Martin; Stulík, Jiří; Novak, Jan; Renfrow, Matthew B; Šebela, Marek

2013-10-30

Determining disease-associated changes in protein glycosylation provides a better understanding of pathogenesis. This work focuses on human immunoglobulin A1 (IgA1), where aberrant O-glycosylation plays a key role in the pathogenesis of IgA nephropathy (IgAN). Normal IgA1 hinge region carries 3 to 6 O-glycans consisting of N-acetylgalactosamine (GalNAc) and galactose (Gal); both sugars may be sialylated. In IgAN patients, some O-glycans on a fraction of IgA1 molecules are Gal-deficient. Here we describe a sample preparation protocol with optimized cysteine alkylation of a Gal-deficient polymeric IgA1 myeloma protein prior to in-gel digestion and analysis of the digest by MALDI-TOF/TOF mass spectrometry (MS). Following a novel strategy, IgA1 hinge-region O-glycopeptides were fractionated by reversed-phase liquid chromatography using a microgradient device and identified by MALDI-TOF/TOF tandem MS (MS/MS). The acquired MS/MS spectra were interpreted manually and by means of our own software. This allowed assigning up to six O-glycosylation sites and demonstration, for the first time, of the distribution of isomeric O-glycoforms having the same molecular mass, but a different glycosylation pattern. The most abundant Gal-deficient O-glycoforms were GalNAc4Gal3 and GalNAc5Gal4 with one Gal-deficient site and GalNAc5Gal3 and GalNAc4Gal2 with two Gal-deficient sites. The most frequent Gal-deficient sites were at Ser230 and/or Thr236. In this work, we studied the O-glycosylation in the hinge region of human immunoglobulin A1 (IgA1). Aberrant glycosylation of the protein plays a key role in the pathogenesis of IgA nephropathy. Thus identification of the O-glycan composition of IgA1 is important for a deeper understanding of the disease mechanism, biomarker discovery and validation, and implementation and monitoring of disease-specific therapies. We developed a new procedure for elucidating the heterogeneity of IgA1 O-glycosylation. After running a polyacrylamide gel

Chemical Modification of Radiation Copolymerized of [N-Vinyl-2-Pyrrolidone/2-(4-Methoxy-Benzylidine)-Malononitrile] with Some Organic Compounds and Their Biological Activity

International Nuclear Information System (INIS)

El Sawy, N.M.; Mostafa, T.B.

2012-01-01

N-Vinyl-2-Pyrrolidone (NVP) was copolymerized with 2-(4-methoxy benzylidine)-malononitrile (MBM) monomers to form a new copolymer, P(MBM-co-NVP), containing nitrile groups. The characterization of copolymer and their modified with organic compounds such as thiourea, thiosemicarbazide,2-aminothiazole, N-Glycyl glycine, 4-amino-N-[5-methyl-3-isoxazolyl]benzene sulfonamide and 4-amino-N-[4-methyl-2-pyrimidnyl]benzene sulfonamide was carried out by Fourier transform infrared spectroscopy (FTIR), Elemental analysis (EA), Gel Permeation Chromatograph (GPC), Differential Scanning Calorimetry (DSC) and Thermal Gravimetric analysis (TGA). The changes in surface morphology of the prepared copolymer and its modified copolymers were observed by scanning electron microscopy (SEM). The biological activity of the modified copolymers with some organic compounds containing with or without sulfur was investigated. The results revealed that the biological activity of the modified copolymers was higher than that of the copolymer ones, under the same conditions. An improvement of prepared copolymers by modification with various organic compounds showed great promise in some practical applications in the field of antibacterial activity.

Sensitivity of HIV-1 to neutralization by antibodies against O-linked carbohydrate epitopes despite deletion of O-glycosylation signals in the V3 loop

DEFF Research Database (Denmark)

Hansen, J E; Jansson, B; Gram, G J

1996-01-01

It has been suggested that threonine or serine residues in the V3 loop of HIV-1 gp120 are glycosylated with the short-chain O-linked oligosaccharides Tn or sialosyl-Tn that function as epitopes for broadly neutralizing carbohydrate specific antibodies. In this study we examined whether mutation....... Additionally, one of these T-A mutants (T308A) also abrogated the signal for N-glycosylation at N306 inside the V3-loop. The mutant clones were compared with the wild type virus as to sensitivity to neutralization with monoclonal and polyclonal antibodies specific for the tip of the V3 loop of BRU or for the O......-linked oligosaccharides Tn or sialosyl-Tn. Deletion of the N-linked oligosaccharide at N306 increased the neutralization sensitivity to antibodies specific for the tip of the loop, which indicates that N-linked glycosylation modulates the accessibility to this immunodominant epitope. However, none of the mutants...

Transgenic rice seed expressing flavonoid biosynthetic genes accumulate glycosylated and/or acylated flavonoids in protein bodies

Science.gov (United States)

Ogo, Yuko; Mori, Tetsuya; Nakabayashi, Ryo; Saito, Kazuki; Takaiwa, Fumio

2016-01-01

Plant-specialized (or secondary) metabolites represent an important source of high-value chemicals. In order to generate a new production platform for these metabolites, an attempt was made to produce flavonoids in rice seeds. Metabolome analysis of these transgenic rice seeds using liquid chromatography-photodiode array-quadrupole time-of-flight mass spectrometry was performed. A total of 4392 peaks were detected in both transgenic and non-transgenic rice, 20–40% of which were only detected in transgenic rice. Among these, 82 flavonoids, including 37 flavonols, 11 isoflavones, and 34 flavones, were chemically assigned. Most of the flavonols and isoflavones were O-glycosylated, while many flavones were O-glycosylated and/or C-glycosylated. Several flavonoids were acylated with malonyl, feruloyl, acetyl, and coumaroyl groups. These glycosylated/acylated flavonoids are thought to have been biosynthesized by endogenous rice enzymes using newly synthesized flavonoids whose biosynthesis was catalysed by exogenous enzymes. The subcellular localization of the flavonoids differed depending on the class of aglycone and the glycosylation/acylation pattern. Therefore, flavonoids with the intended aglycones were efficiently produced in rice seeds via the exogenous enzymes introduced, while the flavonoids were variously glycosylated/acylated by endogenous enzymes. The results suggest that rice seeds are useful not only as a production platform for plant-specialized metabolites such as flavonoids but also as a tool for expanding the diversity of flavonoid structures, providing novel, physiologically active substances. PMID:26438413

The glycosylation status of the murine hepatitis coronavirus M protein affects the interferogenic capacity of the virus in vitro and its ability to replicate in the liver but not the brain

International Nuclear Information System (INIS)

Haan, Cornelis A.M. de; Wit, Marel de; Kuo, Lili; Montalto-Morrison, Cynthia; Haagmans, Bart L.; Weiss, Susan R.; Masters, Paul S.; Rottier, Peter J.M.

2003-01-01

The coronavirus M protein, the most abundant coronaviral envelope component, is invariably glycosylated, which provides the virion with a diffuse, hydrophilic cover on its outer surface. Remarkably, while the group 1 and group 3 coronaviruses all have M proteins with N-linked sugars, the M proteins of the group 2 coronaviruses [e.g., mouse hepatitis virus (MHV)] are O-glycosylated. The conservation of the N- and O-glycosylation motifs suggests that each of these types of carbohydrate modifications is beneficial to their respective virus. Since glycosylation of the M protein is not required for virus assembly, the oligosaccharides are likely to be involved in the virus-host interaction. In order to investigate the role of the M protein glycosylation in the host, two genetically modified MHVs were generated by using targeted RNA recombination. The recombinant viruses carried M proteins that were either N-glycosylated or not glycosylated at all, and these were compared with the parental, O-glycosylated, virus. The M protein glycosylation state did not influence the tissue culture growth characteristics of the recombinant viruses. However, it affected their interferogenic capacity as measured using fixed, virus-infected cells. Viruses containing M proteins with N-linked sugars induced type I interferons to higher levels than viruses carrying M proteins with O-linked sugars. MHV with unglycosylated M proteins appeared to be a poor interferon inducer. In mice, the recombinant viruses differed in their ability to replicate in the liver, but not in the brain, whereas their in vivo interferogenic capacity did not appear to be affected by their glycosylation status. Strikingly, their abilities to replicate in the liver correlated with their in vitro interferogenic capacity. This apparent correlation might be explained by the functioning of lectins, such as the mannose receptor, which are abundantly expressed in the liver but also play a role in the induction of interferon

A novel mass spectrometric strategy "BEMAP" reveals Extensive O-linked protein glycosylation in Enterotoxigenic Escherichia coli

DEFF Research Database (Denmark)

Boysen, Anders; Palmisano, Giuseppe; Krogh, Thøger Jensen

2016-01-01

The attachment of sugars to proteins via side-chain oxygen atoms (O-linked glycosylation) is seen in all three domains of life. However, a lack of widely-applicable analytical tools has restricted the study of this process, particularly in bacteria. In E. coli, only four O-linked glycoproteins have...... previously been characterized. Here we present a glycoproteomics technique, termed BEMAP, which is based on the beta-elimination of O-linked glycans followed by Michael-addition of a phosphonic acid derivative, and subsequent titanium dioxide enrichment. This strategy allows site-specific mass......-spectrometric identification of proteins with O-linked glycan modifications in a complex biological sample. Using BEMAP we identified cell surface-associated and membrane vesicle glycoproteins from Enterotoxigenic E. coli (ETEC) and non-pathogenic E. coli K-12. We identified 618 glycosylated Serine and Threonine residues...

Role of one N-linked oligosaccharide chain on canine herpesvirus gD in its biological activity.

Science.gov (United States)

Maeda, K; Yokoyama, N; Fujita, K; Xuan, X; Mikami, T

1997-12-01

The YP11mu strain of a plaque-selected canine herpesvirus (CHV) encoded a smaller molecular weight (MW) of gD than those of other strains including YP2 strain (Xuan et al., 1990). When nucleotide sequence of the mutated gD of YP11mu strain (gD(YP11mu)) was compared with that of gDs of other CHV strains, gD(YP11mu) lacked 12 nucleotides encoding 4 amino acids, NKTI, including one predicted potential N-linked glycosylation site and no other change was found in other regions. When the gD(YP11mu) and gD of YP2 strain (gD(YP2)) expressed in COS-7 and insect (Spodoptera frugiperda; Sf9) cells were compared each other, both gDs reacted with a panel of monoclonal antibodies (MAbs) against CHV gD by indirect immunofluorescence analysis and the gD(YP11mu) possessed an MW of approximately 47-51 and 39-44 kDa in COS-7 and Sf9 cells, respectively, which were smaller than the expressed gD(YP2) (approximately 51-55 and 41-46 kDa, respectively) by immunoblot analysis. After treatment with tunicamycin, the MW of both gDs in Sf9 cells became approximately 37 kDa. When hemagglutination (HA) test using canine red blood cells (RBC) were carried out, lysates of Sf9 cells expressing CHV gDs agglutinated canine RBC. Serum from mice inoculated with lysates of Sf9 cells expressing the gDs possessed a high titer of virus-neutralizing (VN) activities against CHV. These results indicated that the deletion of 4 amino acids possessing approximately 4 kDa of glyco-chain from gD of CHV in mammalian cells does not affect HA activity and VN antibody-inducing activity and that this deletion of gD(YP11mu) might be a good selective marker for development of recombinant viruses as a live vaccine.

Blood pressure reduction due to hemoglobin glycosylation in type 2 diabetic patients

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Pedro Cabrales

2008-08-01

Full Text Available Pedro Cabrales1, Miguel A Salazar Vázquez2,3, Beatriz Y Salazar Vázquez3,4, Martha Rodríguez-Morán5, Marcos Intaglietta4, Fernando Guerrero-Romero51La Jolla Bioengineering Institute, La Jolla, California, USA; 2Hospital Regional No. 1, of the Mexican Social Security Institute, Victoria de Durango, Dgo. Mexico; 3Faculty of Medicine and Dept. of Physical Chemistry, Universidad Juárez del Estado de Durango, Victoria de Durango, Dgo. Mexico; 4Department of Bioengineering, University of California, San Diego, La Jolla, California, USA; 5Biomedical Research Unit, of the Mexican Social Security Institute, Victoria de Durango, Dgo. MexicoObjective: To test the hypothesis that glycosylation of hemoglobin constitutes a risk factor for hypertension.Methods: A total of 129 relative uniform diabetic subjects (86 women and 42 men were enrolled in a cross-sectional study. Exclusion criteria included alcohol consumption, smoking, ischemic heart disease, stroke, neoplasia, renal, hepatic, and chronic inflammatory disease. Systolic and diastolic pressures were recorded in subsequent days and mean arterial blood pressure (MAP was determined. Hemoglobin glycosylation was measured by determining the percentage glycosylated hemoglobin (HbA1c by means of the automated microparticle enzyme immunoassay test.Results: MAP was found to be independent of the concentration of HbA1c; however, correcting MAP for the variability in hematocrit, to evidence the level of vasoconstriction (or vasodilatation showed that MAP is negatively correlated with the concentration of HbA1c (p for trend <0.05, when patients treated for hypertension are excluded from the analysis. Patients treated for hypertension showed the opposite trend with increasing MAP as HbA1c increased (p for the difference in trends <0.05.Conclusions: Glycosylation per se appears to lead to blood pressure reduction in type 2 diabetic patients untreated for hypertension. Treatment for hypertension may be

Towards the implementation of quality by design to the production of therapeutic monoclonal antibodies with desired glycosylation patterns.

Science.gov (United States)

del Val, Ioscani Jimenez; Kontoravdi, Cleo; Nagy, Judit M

2010-01-01

Quality by design (QbD) is a scheme for the development, manufacture, and approval of pharmaceutical products. The end goal of QbD is to ensure product quality by building it into the manufacturing process. The main regulatory bodies are encouraging its implementation to the manufacture of all new pharmaceuticals including biological products. Monoclonal antibodies (mAbs) are currently the leading products of the biopharmaceutical industry. It has been widely reported that glycosylation directly influences the therapeutic mechanisms by which mAbs function in vivo. In addition, glycosylation has been identified as one of the main sources of monoclonal antibody heterogeneity, and thus, a critical parameter to follow during mAb manufacture. This article reviews the research on glycosylation of mAbs over the past 2 decades under the QbD scope. The categories presented under this scope are: (a) definition of the desired clinical effects of mAbs, (b) definition of the glycosylation-associated critical quality attributes (glycCQAs) of mAbs, (c) assessment of process parameters that pose a risk for mAb glycCQAs, and (d) methods for accurately quantifying glycCQAs of mAbs. The information available in all four areas leads us to conclude that implementation of QbD to the manufacture of mAbs with specific glycosylation patterns will be a reality in the near future. We also foresee that the implementation of QbD will lead to the development of more robust and efficient manufacturing processes and to a new generation of mAbs with increased clinical efficacy. Copyright © 2010 American Institute of Chemical Engineers (AIChE).

25-Hydroxycholesterol Inhibition of Lassa Virus Infection through Aberrant GP1 Glycosylation

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Punya Shrivastava-Ranjan

2016-12-01

Full Text Available Lassa virus (LASV infection is a major public health concern due to high fatality rates and limited effective treatment. The interferon-stimulated gene cholesterol 25-hydroxylase (CH25H encodes an enzyme that catalyzes the production of 25-hydroxycholesterol (25HC. 25HC is involved in regulating cholesterol biosynthesis and has recently been identified as a potent antiviral targeting enveloped virus entry. Here, we show a previously unrecognized role of CH25H in inhibiting LASV glycoprotein glycosylation and the production of infectious virus. Overexpression of CH25H or treatment with 25HC decreased LASV G1 glycoprotein N-glycan maturation and reduced the production of infectious LASV. Depletion of endogenous CH25H using small interfering RNA (siRNA enhanced the levels of fully glycosylated G1 and increased infectious LASV production. Finally, LASV particles produced from 25HC-treated cells were found to be less infectious, to incorporate aberrantly glycosylated GP1 species, and to be defective in binding alpha-dystroglycan, an attachment and entry receptor. Our findings identify a novel role for CH25H in controlling LASV propagation and indicate that manipulation of the expression of CH25H or the administration of 25HC may be a useful anti-LASV therapy.

Investigations on aberrant glycosylation of glycosphingolipids in colorectal cancer tissues using liquid chromatography and matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS).

Science.gov (United States)

Holst, Stephanie; Stavenhagen, Kathrin; Balog, Crina I A; Koeleman, Carolien A M; McDonnell, Liam M; Mayboroda, Oleg A; Verhoeven, Aswin; Mesker, Wilma E; Tollenaar, Rob A E M; Deelder, André M; Wuhrer, Manfred

2013-11-01

Cancer is a leading cause of death and alterations of glycosylation are characteristic features of malignant cells. Colorectal cancer is one of the most common cancers and its exact causes and biology are not yet well understood. Here, we compared glycosylation profiles of colorectal tumor tissues and corresponding control tissues of 13 colorectal cancer patients to contribute to the understanding of this cancer. Using MALDI-TOF(/TOF)-MS and 2-dimensional LC-MS/MS we characterized enzymatically released and 2-aminobenzoic acid labeled glycans from glycosphingolipids. Multivariate data analysis revealed significant differences between tumor and corresponding control tissues. Main discriminators were obtained, which represent the overall alteration in glycosylation of glycosphingolipids during colorectal cancer progression, and these were found to be characterized by (1) increased fucosylation, (2) decreased acetylation, (3) decreased sulfation, (4) reduced expression of globo-type glycans, as well as (5) disialyl gangliosides. The findings of our current research confirm former reports, and in addition expand the knowledge of glycosphingolipid glycosylation in colorectal cancer by revealing new glycans with discriminative power and characteristic, cancer-associated glycosylation alterations. The obtained discriminating glycans can contribute to progress the discovery of biomarkers to improve diagnostics and patient treatment.

Investigations on Aberrant Glycosylation of Glycosphingolipids in Colorectal Cancer Tissues Using Liquid Chromatography and Matrix-Assisted Laser Desorption Time-of-Flight Mass Spectrometry (MALDI-TOF-MS)*

Science.gov (United States)

Holst, Stephanie; Stavenhagen, Kathrin; Balog, Crina I. A.; Koeleman, Carolien A. M.; McDonnell, Liam M.; Mayboroda, Oleg A.; Verhoeven, Aswin; Mesker, Wilma E.; Tollenaar, Rob A. E. M.; Deelder, André M.; Wuhrer, Manfred

2013-01-01

Cancer is a leading cause of death and alterations of glycosylation are characteristic features of malignant cells. Colorectal cancer is one of the most common cancers and its exact causes and biology are not yet well understood. Here, we compared glycosylation profiles of colorectal tumor tissues and corresponding control tissues of 13 colorectal cancer patients to contribute to the understanding of this cancer. Using MALDI-TOF(/TOF)-MS and 2-dimensional LC-MS/MS we characterized enzymatically released and 2-aminobenzoic acid labeled glycans from glycosphingolipids. Multivariate data analysis revealed significant differences between tumor and corresponding control tissues. Main discriminators were obtained, which represent the overall alteration in glycosylation of glycosphingolipids during colorectal cancer progression, and these were found to be characterized by (1) increased fucosylation, (2) decreased acetylation, (3) decreased sulfation, (4) reduced expression of globo-type glycans, as well as (5) disialyl gangliosides. The findings of our current research confirm former reports, and in addition expand the knowledge of glycosphingolipid glycosylation in colorectal cancer by revealing new glycans with discriminative power and characteristic, cancer-associated glycosylation alterations. The obtained discriminating glycans can contribute to progress the discovery of biomarkers to improve diagnostics and patient treatment. PMID:23878401

Analytical tools for the study of cellular glycosylation in the immune system

Directory of Open Access Journals (Sweden)

Yvette eVan Kooyk

2013-12-01

Full Text Available It is becoming increasingly clear that glycosylation plays important role in intercellular communication within the immune system. Glycosylation-dependent interactions are crucial for the innate and adaptive immune system and regulate immune cell trafficking, synapse formation, activation, and survival. These functions take place by the cis or trans interaction of lectins with glycans. Classical immunological and biochemical methods have been used for the study of lectin function; however, the investigation of their counterparts, glycans, requires very specialized methodologies that have been extensively developed in the past decade within the Glycobiology scientific community. This Mini-Review intends to summarize the available technology for the study of glycan biosynthesis, its regulation and characterization for their application to the study of glycans in Immunology.

Neuronal glycosylation differentials in normal, injured and chondroitinase-treated environments

International Nuclear Information System (INIS)

Kilcoyne, Michelle; Sharma, Shashank; McDevitt, Niamh; O’Leary, Claire; Joshi, Lokesh; McMahon, Siobhán S.

2012-01-01

Highlights: ► Carbohydrates are important in the CNS and ChABC has been used for spinal cord injury (SCI) treatment. ► Neuronal glycosylation in injury and after ChABC treatment is unknown. ► In silico mining verified that glyco-related genes were differentially regulated after SCI. ► In vitro model system revealed abnormal sialylation in an injured environment. ► The model indicated a return to normal neuronal glycosylation after ChABC treatment. -- Abstract: Glycosylation is found ubiquitously throughout the central nervous system (CNS). Chondroitin sulphate proteoglycans (CSPGs) are a group of molecules heavily substituted with glycosaminoglycans (GAGs) and are found in the extracellular matrix (ECM) and cell surfaces. Upon CNS injury, a glial scar is formed, which is inhibitory for axon regeneration. Several CSPGs are up-regulated within the glial scar, including NG2, and these CSPGs are key inhibitory molecules of axonal regeneration. Treatment with chondroitinase ABC (ChABC) can neutralise the inhibitory nature of NG2. A gene expression dataset was mined in silico to verify differentially regulated glycosylation-related genes in neurons after spinal cord injury and identify potential targets for further investigation. To establish the glycosylation differential of neurons that grow in a healthy, inhibitory and ChABC-treated environment, we established an indirect co-culture system where PC12 neurons were grown with primary astrocytes, Neu7 astrocytes (which overexpress NG2) and Neu7 astrocytes treated with ChABC. After 1, 4 and 8 days culture, lectin cytochemistry of the neurons was performed using five fluorescently-labelled lectins (ECA MAA, PNA, SNA-I and WFA). Usually α-(2,6)-linked sialylation scarcely occurs in the CNS but this motif was observed on the neurons in the injured environment only at day 8. Treatment with ChABC was successful in returning neuronal glycosylation to normal conditions at all timepoints for MAA, PNA and SNA-I staining

Neuronal glycosylation differentials in normal, injured and chondroitinase-treated environments

Energy Technology Data Exchange (ETDEWEB)

Kilcoyne, Michelle; Sharma, Shashank [Glycoscience Group, National Centre for Biomedical Engineering Science, National University of Ireland, Galway (Ireland); McDevitt, Niamh; O' Leary, Claire [Anatomy, School of Medicine, National University of Ireland, Galway (Ireland); Joshi, Lokesh [Glycoscience Group, National Centre for Biomedical Engineering Science, National University of Ireland, Galway (Ireland); McMahon, Siobhan S., E-mail: siobhan.mcmahon@nuigalway.ie [Anatomy, School of Medicine, National University of Ireland, Galway (Ireland)

2012-04-13

Highlights: Black-Right-Pointing-Pointer Carbohydrates are important in the CNS and ChABC has been used for spinal cord injury (SCI) treatment. Black-Right-Pointing-Pointer Neuronal glycosylation in injury and after ChABC treatment is unknown. Black-Right-Pointing-Pointer In silico mining verified that glyco-related genes were differentially regulated after SCI. Black-Right-Pointing-Pointer In vitro model system revealed abnormal sialylation in an injured environment. Black-Right-Pointing-Pointer The model indicated a return to normal neuronal glycosylation after ChABC treatment. -- Abstract: Glycosylation is found ubiquitously throughout the central nervous system (CNS). Chondroitin sulphate proteoglycans (CSPGs) are a group of molecules heavily substituted with glycosaminoglycans (GAGs) and are found in the extracellular matrix (ECM) and cell surfaces. Upon CNS injury, a glial scar is formed, which is inhibitory for axon regeneration. Several CSPGs are up-regulated within the glial scar, including NG2, and these CSPGs are key inhibitory molecules of axonal regeneration. Treatment with chondroitinase ABC (ChABC) can neutralise the inhibitory nature of NG2. A gene expression dataset was mined in silico to verify differentially regulated glycosylation-related genes in neurons after spinal cord injury and identify potential targets for further investigation. To establish the glycosylation differential of neurons that grow in a healthy, inhibitory and ChABC-treated environment, we established an indirect co-culture system where PC12 neurons were grown with primary astrocytes, Neu7 astrocytes (which overexpress NG2) and Neu7 astrocytes treated with ChABC. After 1, 4 and 8 days culture, lectin cytochemistry of the neurons was performed using five fluorescently-labelled lectins (ECA MAA, PNA, SNA-I and WFA). Usually {alpha}-(2,6)-linked sialylation scarcely occurs in the CNS but this motif was observed on the neurons in the injured environment only at day 8. Treatment

DISAL glycosyl donors for the synthesis of a linear hexasaccharide under mild conditions

DEFF Research Database (Denmark)

Petersen, Lars; Laursen, Jane B.; Larsen, K.

2003-01-01

The new class of glycosyl donors with a methyl 3,5-dinitrosalicylate (DISAL) anomeric leaving group has proved efficient for glycosylation under strictly neutral, mildly basic, or mildly acidic conditions. Here, we report the synthesis of novel DISAL disaccharide glycosyl donors prepared by easy...... nucleophilic aromatic substitution. These DISAL donors proved efficient in the synthesis of a starch-related hexasaccharide under very mild conditions. Glycosylations proceeded with alpha-selectivity and were compatible with Trt protecting groups....

DNA methylation in Cosmc promoter region and aberrantly glycosylated IgA1 associated with pediatric IgA nephropathy.

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Qiang Sun

Full Text Available IgA nephropathy (IgAN is one of the most common glomerular diseases leading to end-stage renal failure. Elevation of aberrantly glycosylated IgA1 is a key feature of it. The expression of the specific molecular chaperone of core1ß1, 3galactosyl transferase (Cosmc is known to be reduced in IgAN. We aimed to investigate whether the methylation of CpG islands of Cosmc gene promoter region could act as a possible mechanism responsible for down-regulation of Cosmc and related higher secretion of aberrantly glycosylated IgA1in lymphocytes from children with IgA nephropathy. Three groups were included: IgAN children (n = 26, other renal diseases (n = 11 and healthy children (n = 13. B-lymphocytes were isolated and cultured, treated or not with IL-4 or 5-Aza-2'-deoxycytidine (AZA. The levels of DNA methylation of Cosmc promotor region were not significantly different between the lymphocytes of the three children populations (P = 0.113, but there were significant differences between IgAN lymphocytes and lymphocytes of the other two children populations after IL-4 (P<0.0001 or AZA (P<0.0001. Cosmc mRNA expression was low in IgAN lymphocytes compared to the other two groups (P<0.0001. The level of aberrantly glycosylated IgA1 was markedly higher in IgAN group compared to the other groups (P<0.0001. After treatment with IL-4, the levels of Cosmc DNA methylation and aberrantly glycosylated IgA1 in IgAN lymphocytes were remarkably higher than the other two groups (P<0.0001 with more markedly decreased Cosmc mRNA content (P<0.0001. After treatment with AZA, the levels in IgAN lymphocytes were decreased, but was still remarkably higher than the other two groups (P<0.0001, while Cosmc mRNA content in IgAN lymphocytes were more markedly increased than the other two groups (P<0.0001. The alteration of DNA methylation by IL-4 or AZA specifically correlates in IgAN lymphocytes with alterations in Cosmc mRNA expression and with the level of aberrantly glycosylated

Location, location, location: new insights into O-GalNAc protein glycosylation

DEFF Research Database (Denmark)

Gill, David J; Clausen, Henrik; Bard, Frederic

2011-01-01

O-GalNAc glycosylation of proteins confers essential structural, protective and signaling roles in eumetazoans. Addition of O-glycans onto proteins is an extremely complex process that regulates both sites of attachment and the types of oligosaccharides added. Twenty distinct polypeptide GalNAc......-transferases (GalNAc-Ts) initiate O-glycosylation and fine-tuning their expression provides a mechanism for regulating this action. Recently, a new mode of regulation has emerged where activation of Src kinase selectively redistributes Golgi-localized GalNAc-Ts to the ER. This relocalization results in a strong...... increase in the density of O-glycan decoration. In this review, we discuss how different mechanisms can regulate the number and the types of O-glycans decorating proteins. In addition, we speculate how Src-dependent relocation of GalNAc-Ts could play an important role in cancerous cellular transformation....

Strategies toward protecting group-free glycosylation through selective activation of the anomeric center

Czech Academy of Sciences Publication Activity Database

Downey, Alan Michael; Hocek, Michal

2017-01-01

Roč. 13, Jun 27 (2017), s. 1239-1279 ISSN 1860-5397 R&D Projects: GA TA ČR(CZ) TE01020028 Grant - others:AV ČR(CZ) AP1501 Program:Akademická prémie - Praemium Academiae Institutional support: RVO:61388963 Keywords : glycosides * glycosylation * oligosaccharides * protecting groups Subject RIV: CC - Organic Chemistry OBOR OECD: Organic chemistry Impact factor: 2.337, year: 2016 http://www.beilstein-journals.org/bjoc/articles/13/123

The Influences of Glycosylation on the Antigenicity, Immunogenicity, and Protective Efficacy of Ebola Virus GP DNA Vaccines

National Research Council Canada - National Science Library

Dowling, William; Thompson, Elizabeth; Badger, Catherine; Mellquist, Jenny L; Garrison, Aura R; Smith, Jeffrey M; Paragas, Jason; Hogan, Robert J; Schmaljohn, Connie

2006-01-01

... or with deletions in the central hypervariable mucin region. We showed that mutation of one of the two N-linked GP2 glycosylation sites was highly detrimental to the antigenicity and immunogenicity of GP...

N-Glycosylation analysis of yeast Carboxypeptidase Y reveals the ultimate removal of phosphate from glycans at Asn368.

Science.gov (United States)

B S, Gnanesh Kumar; Surolia, Avadhesha

2017-05-01

Carboxypeptidase Y from Saccharomyces cerivisiae was characterized for its site specific N-glycosylation through mass spectrometry. The N-glycopeptides were derived using non specific proteases and are analysed directly on liquid chromatography coupled to ion trap mass spectrometer in tandem mode. The evaluation of glycan fragment ions and the Y 1 ions (peptide+HexNAc) +n revealed the glycan sequence and the corresponding site of attachment. We observed the microheterogeneity in N-glycans such as Man 11-15 GlcNAc 2 at Asn 13 , Man 8-12 GlcNAc 2 at Asn 87 , Man 9-14 GlcNAc 2 at Asn 168 and phosphorylated Man 12-17 GlcNAc 2 as well as Man 11-16 GlcNAc 2 at Asn 368 . The presence of N-glycans with Man <18 GlcNAc 2 indicated that in vacuoles the steady release of mannose/phospho mannose residues from glycans occurs initially at Asn 13 or Asn 168 followed by at Asn 368 . However, glycans at Asn 87 which comprises Man 8-12 residues as reported earlier remain intact suggesting its inaccessibility for a similar processing. This in turn indicates the interaction of the glycan at Asn 87 with the polypeptide chain implicating it in the folding of the protein. Copyright © 2017 Elsevier B.V. All rights reserved.

Nonenzymatic glycosylation of bovine myelin basic protein

International Nuclear Information System (INIS)

Hitz, J.B.

1987-01-01

In the CNS myelin sheath the nonenzymatic glycosylation reaction (at the early stage of the Amadori product) occurs only with the myelin basic protein and not with the other myelin proteins. This was observed in isolated bovine myelin by in vitro incubation with [ 14 C]-galactose and [ 14 C]-glucose. The respective in-vitro incorporation rates for purified bovine myelin basic protein with D-galactose, D-glucose and D-mannose were 7.2, 2.4 and 2.4 mmoles/mole myelin basic protein per day at 37 0 C. A more rapid, HPLC method was devised and characterized to specifically analyze for the Amadori product. The HPLC method was correlated to the [ 14 C]-sugar incorporation method for myelin basic protein under a set of standard reaction conditions using [ 14 C]-glucose and [ 14 C]-mannose with HPLC values at 1/6 and 1/5 of the [ 14 C]-sugar incorporation method. A novel myelin basic protein purification step has been developed that yields a relativity proteolytic free preparation that is easy to work with, being totally soluble at a neutral pH. Nine new spots appear for a trypsinized glycosylated MBP in the paper peptide map of which eight correspond to positions of the [ 3 H]-labeled Amadori product in affinity isolated peptides. These studies provide a general characterization of and a structural basis for investigations on nonenzymatically glycosylated MBP as well as identifying MBP as the only nonenzymatically glycosylated protein in the CNS myelin sheath which may accumulate during aging, diabetes, and demyelinating diseases in general

N-linked (N-) glycoproteomics of urinary exosomes. [Corrected].

Science.gov (United States)

Saraswat, Mayank; Joenväära, Sakari; Musante, Luca; Peltoniemi, Hannu; Holthofer, Harry; Renkonen, Risto

2015-02-01

Epithelial cells lining the urinary tract secrete urinary exosomes (40-100 nm) that can be targeted to specific cells modulating their functionality. One potential targeting mechanism is adhesion between vesicle surface glycoproteins and target cells. This makes the glycopeptide analysis of exosomes important. Exosomes reflect the physiological state of the parent cells; therefore, they are a good source of biomarkers for urological and other diseases. Moreover, the urine collection is easy and noninvasive and urinary exosomes give information about renal and systemic organ systems. Accordingly, multiple studies on proteomic characterization of urinary exosomes in health and disease have been published. However, no systematic analysis of their glycoproteomic profile has been carried out to date, whereas a conserved glycan signature has been found for exosomes from urine and other sources including T cell lines and human milk. Here, we have enriched and identified the N-glycopeptides from these vesicles. These enriched N-glycopeptides were solved for their peptide sequence, glycan composition, structure, and glycosylation site using collision-induced dissociation MS/MS (CID-tandem MS) data interpreted by a publicly available software GlycopeptideId. Released glycans from the same sample was also analyzed with MALDI-MS. We have identified the N-glycoproteome of urinary exosomes. In total 126 N-glycopeptides from 51 N-glycosylation sites belonging to 37 glycoproteins were found in our results. The peptide sequences of these N-glycopeptides were identified unambiguously and their glycan composition (for 125 N-glycopeptides) and structures (for 87 N-glycopeptides) were proposed. A corresponding glycomic analysis with released N-glycans was also performed. We identified 66 unique nonmodified N-glycan compositions and in addition 13 sulfated/phosphorylated glycans were also found. This is the first systematic analysis of N-glycoproteome of urinary exosomes. © 2015 by The

Charged particle activation analysis of phosphorus in biological materials

International Nuclear Information System (INIS)

Masumoto, K.; Yagi, M.

1983-01-01

Charged particle activation analysis of phosphorus in biological materials using the 31 P(α,n) sup(34m)Cl reaction has been studied. Since sup(34m)Cl is also produced by the 32 S(α,pn) and the 35 Cl(α,α'n) reactions, the thick-target yield curves on phosphorus, sulfur and chlorine were determined in order to choose the optimum irradiation conditions. As a result, it was found that the activation analysis for phosphorus without interferences from sulfur and chlorine is possible by bombarding with less than 17 MeV alphas. The applicability of this method to biological samples was then examined by irradiating several standard reference materials. It was confirmed that phosphorus can readily be determined at the detection limit of 1μg free from interferences due to the matrix elements. (author)

Human IGF-I propeptide A promotes articular chondrocyte biosynthesis and employs glycosylation-dependent heparin binding.

Science.gov (United States)

Shi, Shuiliang; Kelly, Brian J; Wang, Congrong; Klingler, Ken; Chan, Albert; Eckert, George J; Trippel, Stephen B

2018-03-01

Insulin-like growth factor I (IGF-I) is a key regulator of chondrogenesis, but its therapeutic application to articular cartilage damage is limited by rapid elimination from the repair site. The human IGF-I gene gives rise to three IGF-I propeptides (proIGF-IA, proIGF-IB and proIGF-IC) that are cleaved to create mature IGF-I. In this study, we elucidate the processing of IGF-I precursors by articular chondrocytes, and test the hypotheses that proIGF-I isoforms bind to heparin and regulate articular chondrocyte biosynthesis. Human IGF-I propeptides and mutants were overexpressed in bovine articular chondrocytes. IGF-I products were characterized by ELISA, western blot and FPLC using a heparin column. The biosynthetic activity of IGF-I products on articular chondrocytes was assayed for DNA and glycosaminoglycan that the cells produced. Secreted IGF-I propeptides stimulated articular chondrocyte biosynthetic activity to the same degree as mature IGF-I. Of the three IGF-I propeptides, only one, proIGF-IA, strongly bound to heparin. Interestingly, heparin binding of proIGF-IA depended on N-glycosylation at Asn92 in the EA peptide. To our knowledge, this is the first demonstration that N-glycosylation determines the binding of a heparin-binding protein to heparin. The biosynthetic and heparin binding abilities of proIGF-IA, coupled with its generation of IGF-I, suggest that proIGF-IA may have therapeutic value for articular cartilage repair. These data identify human pro-insulin-like growth factor IA as a bifunctional protein. Its combined ability to bind heparin and augment chondrocyte biosynthesis makes it a promising therapeutic agent for cartilage damage due to trauma and osteoarthritis. Copyright © 2017 Elsevier B.V. All rights reserved.

Engineering the yeast Yarrowia lipolytica for the production of therapeutic proteins homogeneously glycosylated with Man8GlcNAc2 and Man5GlcNAc2

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De Pourcq Karen

2012-05-01

Full Text Available Abstract Background Protein-based therapeutics represent the fastest growing class of compounds in the pharmaceutical industry. This has created an increasing demand for powerful expression systems. Yeast systems are widely used, convenient and cost-effective. Yarrowia lipolytica is a suitable host that is generally regarded as safe (GRAS. Yeasts, however, modify their glycoproteins with heterogeneous glycans containing mainly mannoses, which complicates downstream processing and often interferes with protein function in man. Our aim was to glyco-engineer Y. lipolytica to abolish the heterogeneous, yeast-specific glycosylation and to obtain homogeneous human high-mannose type glycosylation. Results We engineered Y. lipolytica to produce homogeneous human-type terminal-mannose glycosylated proteins, i.e. glycosylated with Man8GlcNAc2 or Man5GlcNAc2. First, we inactivated the yeast-specific Golgi α-1,6-mannosyltransferases YlOch1p and YlMnn9p; the former inactivation yielded a strain producing homogeneous Man8GlcNAc2 glycoproteins. We tested this strain by expressing glucocerebrosidase and found that the hypermannosylation-related heterogeneity was eliminated. Furthermore, detailed analysis of N-glycans showed that YlOch1p and YlMnn9p, despite some initial uncertainty about their function, are most likely the α-1,6-mannosyltransferases responsible for the addition of the first and second mannose residue, respectively, to the glycan backbone. Second, introduction of an ER-retained α-1,2-mannosidase yielded a strain producing proteins homogeneously glycosylated with Man5GlcNAc2. The use of the endogenous LIP2pre signal sequence and codon optimization greatly improved the efficiency of this enzyme. Conclusions We generated a Y. lipolytica expression platform for the production of heterologous glycoproteins that are homogenously glycosylated with either Man8GlcNAc2 or Man5GlcNAc2 N-glycans. This platform expands the utility of Y. lipolytica as a

Therapeutic monoclonal antibody N-glycosylation - Structure, function and therapeutic potential.

Science.gov (United States)

Cymer, Florian; Beck, Hermann; Rohde, Adelheid; Reusch, Dietmar

2018-03-01

Therapeutic antibodies (IgG-type) contain several post-translational modifications (PTMs) whereby introducing a large heterogeneity, both structural and functional, into this class of therapeutics. Of these modifications, glycosylation in the fragment crystallizable (Fc) region is the most heterogeneous PTM, which can affect the stability of the molecule and interactions with Fc-receptors in vivo. Hence, the glycoform distribution can affect the mode of action and have implications for bioactivity, safety and efficacy of the drug. Main topics of the manuscript include: What factors influence the (Fc) glycan pattern in therapeutic antibodies and how can these glycans be characterized? How does structure of the Fc-glycan relate to function and what methods are available to characterize those functions? Although heterogeneous in their scope, the different sections are intended to combine current knowledge on structure-function correlations of IgG glycan structures with regard to Fc (effector) functions, as well as basic aspects and methodologies for their assessment. Copyright © 2017. Published by Elsevier Ltd.

Cancer associated aberrant protein o-glycosylation can modify antigen processing and immune response

DEFF Research Database (Denmark)

Madsen, Caroline B; Petersen, Cecilie; Lavrsen, Kirstine

2012-01-01

Aberrant glycosylation of mucins and other extracellular proteins is an important event in carcinogenesis and the resulting cancer associated glycans have been suggested as targets in cancer immunotherapy. We assessed the role of O-linked GalNAc glycosylation on antigen uptake, processing......, and presentation on MHC class I and II molecules. The effect of GalNAc O-glycosylation was monitored with a model system based on ovalbumin (OVA)-MUC1 fusion peptides (+/- glycosylation) loaded onto dendritic cells co-cultured with IL-2 secreting OVA peptide-specific T cell hybridomas. To evaluate the in vivo...

Investigating Spectrum of Biological Activity of 4- and 5-Chloro-2-hydroxy-N-[2-(arylamino-1-alkyl-2-oxoethyl]benzamides

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Jirina Stolarikova

2011-03-01

Full Text Available In this study, a series of twenty-two 5-chloro-2-hydroxy-N-[2-(arylamino-1-alkyl-2-oxoethyl]benzamides and ten 4-chloro-2-hydroxy-N-[2-(arylamino-1-alkyl-2-oxoethyl]benzamides is described. The compounds were analyzed using RP-HPLC to determine lipophilicity. Primary in vitro screening of the synthesized compounds was performed against mycobacterial, bacterial and fungal strains. They were also evaluated for their activity related to the inhibition of photosynthetic electron transport (PET in spinach (Spinacia oleracea L. chloroplasts. The compounds showed biological activity comparable with or higher than the standards isoniazid, fluconazole, penicillin G or ciprofloxacin. For all the compounds, the relationships between the lipophilicity and the chemical structure of the studied compounds as well as their structure-activity relationships are discussed.

Rapid phenolic O-glycosylation of small molecules and complex unprotected peptides in aqueous solvent

Science.gov (United States)

Wadzinski, Tyler J.; Steinauer, Angela; Hie, Liana; Pelletier, Guillaume; Schepartz, Alanna; Miller, Scott J.

2018-06-01

Glycosylated natural products and synthetic glycopeptides represent a significant and growing source of biochemical probes and therapeutic agents. However, methods that enable the aqueous glycosylation of endogenous amino acid functionality in peptides without the use of protecting groups are scarce. Here, we report a transformation that facilitates the efficient aqueous O-glycosylation of phenolic functionality in a wide range of small molecules, unprotected tyrosine, and tyrosine residues embedded within a range of complex, fully unprotected peptides. The transformation, which uses glycosyl fluoride donors and is promoted by Ca(OH)2, proceeds rapidly at room temperature in water, with good yields and selective formation of unique anomeric products depending on the stereochemistry of the glycosyl donor. High functional group tolerance is observed, and the phenol glycosylation occurs selectively in the presence of virtually all side chains of the proteinogenic amino acids with the singular exception of Cys. This method offers a highly selective, efficient, and operationally simple approach for the protecting-group-free synthesis of O-aryl glycosides and Tyr-O-glycosylated peptides in water.

Exploiting the Campylobacter jejuni protein glycosylation system for glycoengineering vaccines and diagnostic tools directed against brucellosis

Directory of Open Access Journals (Sweden)

Iwashkiw Jeremy A

2012-01-01

Full Text Available Abstract Background Immune responses directed towards surface polysaccharides conjugated to proteins are effective in preventing colonization and infection of bacterial pathogens. Presently, the production of these conjugate vaccines requires intricate synthetic chemistry for obtaining, activating, and attaching the polysaccharides to protein carriers. Glycoproteins generated by engineering bacterial glycosylation machineries have been proposed to be a viable alternative to traditional conjugation methods. Results In this work we expressed the C. jejuni oligosaccharyltansferase (OTase PglB, responsible for N-linked protein glycosylation together with a suitable acceptor protein (AcrA in Yersinia enterocolitica O9 cells. MS analysis of the acceptor protein demonstrated the transfer of a polymer of N-formylperosamine to AcrA in vivo. Because Y. enterocolitica O9 and Brucella abortus share an identical O polysaccharide structure, we explored the application of the resulting glycoprotein in vaccinology and diagnostics of brucellosis, one of the most common zoonotic diseases with over half a million new cases annually. Injection of the glycoprotein into mice generated an IgG response that recognized the O antigen of Brucella, although this response was not protective against a challenge with a virulent B. abortus strain. The recombinant glycoprotein coated onto magnetic beads was efficient in differentiating between naïve and infected bovine sera. Conclusion Bacterial engineered glycoproteins show promising applications for the development on an array of diagnostics and immunoprotective opportunities in the future.

Fluorine-Directed Glycosylation Enables the Stereocontrolled Synthesis of Selective SGLT2 Inhibitors for Type II Diabetes.

Science.gov (United States)

Sadurní, Anna; Kehr, Gerald; Ahlqvist, Marie; Wernevik, Johan; Sjögren, Helena Peilot; Kankkonen, Cecilia; Knerr, Laurent; Gilmour, Ryan

2018-02-26

Inhibition of the sodium-glucose co-transporters (SGLT1 and SGLT2) is a validated strategy to address the increasing prevalence of type II diabetes mellitus. However, achieving selective inhibition of human SGLT1 or SGLT2 remains challenging. Orally available small molecule drugs based on the d-glucose core of the natural product Gliflozin have proven to be clinically effective in this regard, effectively impeding glucose reabsorption. Herein, we disclose the influence of molecular editing with fluorine at the C2 position of the pyranose ring of Phlorizin analogues Remogliflozin Etabonate and Dapagliflozin (Farxiga ® ) to concurrently direct β-selective glycosylation, as is required for biological efficacy, and enhance aspects of the physicochemical profile. Given the abundance of glycosylated pharmaceuticals in diabetes therapy that contain a β-configured d-glucose nucleus, it is envisaged that this strategy may prove to be expansive. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biological properties of purified recombinant HCV particles with an epitope-tagged envelope

Energy Technology Data Exchange (ETDEWEB)

Takahashi, Hitoshi; Akazawa, Daisuke [Department of Virology II, National Institute of Infectious Diseases, Tokyo (Japan); Toray Industries, Inc., Kanagawa (Japan); Kato, Takanobu; Date, Tomoko [Department of Virology II, National Institute of Infectious Diseases, Tokyo (Japan); Shirakura, Masayuki [Department of Virology II, National Institute of Infectious Diseases, Tokyo (Japan); Toray Industries, Inc., Kanagawa (Japan); Nakamura, Noriko; Mochizuki, Hidenori [Toray Industries, Inc., Kanagawa (Japan); Tanaka-Kaneko, Keiko; Sata, Tetsutaro [Department of Pathology, National Institute of Infectious Diseases, Tokyo (Japan); Tanaka, Yasuhito [Department of Clinical Molecular Informative Medicine, Nagoya City University Graduate School of Medicine, Nagoya (Japan); Mizokami, Masashi [Research Center for Hepatitis and Immunology, Kohnodai Hospital, International Medical Center of Japan, Chiba (Japan); Suzuki, Tetsuro [Department of Virology II, National Institute of Infectious Diseases, Tokyo (Japan); Wakita, Takaji, E-mail: wakita@nih.go.jp [Department of Virology II, National Institute of Infectious Diseases, Tokyo (Japan)

2010-05-14

To establish a simple system for purification of recombinant infectious hepatitis C virus (HCV) particles, we designed a chimeric J6/JFH-1 virus with a FLAG (FL)-epitope-tagged sequence at the N-terminal region of the E2 hypervariable region-1 (HVR1) gene (J6/JFH-1/1FL). We found that introduction of an adaptive mutation at the potential N-glycosylation site (E2N151K) leads to efficient production of the chimeric virus. This finding suggests the involvement of glycosylation at Asn within the envelope protein(s) in HCV morphogenesis. To further analyze the biological properties of the purified recombinant HCV particles, we developed a strategy for large-scale production and purification of recombinant J6/JFH-1/1FL/E2N151K. Infectious particles were purified from the culture medium of J6/JFH-1/1FL/E2N151K-infected Huh-7 cells using anti-FLAG affinity chromatography in combination with ultrafiltration. Electron microscopy of the purified particles using negative staining showed spherical particle structures with a diameter of 40-60 nm and spike-like projections. Purified HCV particle-immunization induced both an anti-E2 and an anti-FLAG antibody response in immunized mice. This strategy may contribute to future detailed analysis of HCV particle structure and to HCV vaccine development.

Biological properties of purified recombinant HCV particles with an epitope-tagged envelope

International Nuclear Information System (INIS)

Takahashi, Hitoshi; Akazawa, Daisuke; Kato, Takanobu; Date, Tomoko; Shirakura, Masayuki; Nakamura, Noriko; Mochizuki, Hidenori; Tanaka-Kaneko, Keiko; Sata, Tetsutaro; Tanaka, Yasuhito; Mizokami, Masashi; Suzuki, Tetsuro; Wakita, Takaji

2010-01-01

To establish a simple system for purification of recombinant infectious hepatitis C virus (HCV) particles, we designed a chimeric J6/JFH-1 virus with a FLAG (FL)-epitope-tagged sequence at the N-terminal region of the E2 hypervariable region-1 (HVR1) gene (J6/JFH-1/1FL). We found that introduction of an adaptive mutation at the potential N-glycosylation site (E2N151K) leads to efficient production of the chimeric virus. This finding suggests the involvement of glycosylation at Asn within the envelope protein(s) in HCV morphogenesis. To further analyze the biological properties of the purified recombinant HCV particles, we developed a strategy for large-scale production and purification of recombinant J6/JFH-1/1FL/E2N151K. Infectious particles were purified from the culture medium of J6/JFH-1/1FL/E2N151K-infected Huh-7 cells using anti-FLAG affinity chromatography in combination with ultrafiltration. Electron microscopy of the purified particles using negative staining showed spherical particle structures with a diameter of 40-60 nm and spike-like projections. Purified HCV particle-immunization induced both an anti-E2 and an anti-FLAG antibody response in immunized mice. This strategy may contribute to future detailed analysis of HCV particle structure and to HCV vaccine development.

N-linked glycans do not affect plasma membrane localization of multidrug resistance protein 4 (MRP4) but selectively alter its prostaglandin E2 transport activity.

Science.gov (United States)

Miah, M Fahad; Conseil, Gwenaëlle; Cole, Susan P C

2016-01-22

Multidrug resistance protein 4 (MRP4) is a member of subfamily C of the ATP-binding cassette superfamily of membrane transport proteins. MRP4 mediates the ATP-dependent efflux of many endogenous and exogenous solutes across the plasma membrane, and in polarized cells, it localizes to the apical or basolateral plasma membrane depending on the tissue type. MRP4 is a 170 kDa glycoprotein and here we show that MRP4 is simultaneously N-glycosylated at Asn746 and Asn754. Furthermore, confocal immunofluorescence studies showed that N-glycans do not affect MRP4's apical membrane localization in polarized LLC-PK1 cells or basolateral membrane localization in polarized MDCKI cells. However, vesicular transport assays showed that N-glycans differentially affect MRP4's ability to transport prostaglandin E2, but not estradiol glucuronide. Together these data indicate that N-glycosylation at Asn746 and Asn754 is not essential for plasma membrane localization of MRP4 but cause substrate-selective effects on its transport activity. Copyright © 2015 Elsevier Inc. All rights reserved.

A Novel Strategy for Characterization of Glycosylated Proteins Separated by Gel Electrophoresis

DEFF Research Database (Denmark)

Larsen, Martin; Skottrup, Peter; Enghild, Jan Johannes

Protein glycosylation can be vital for changing the function or physiochemical properties of a protein. Abnormal glycosylation can lead to protein malfunction, resulting in severe diseases. Therefore, it is important to develop techniques for characterization of such modifications in proteins...... graphite powder micro-columns in combination with mass spectrometry. The method is faster and more sensitive than previous approaches and would be ideal for proteomics studies and verification of correct glycosylation of recombinant glycoproteins....

Lysosomal enzyme delivery by ICAM-1-targeted nanocarriers bypassing glycosylation- and clathrin-dependent endocytosis.

Science.gov (United States)

Muro, Silvia; Schuchman, Edward H; Muzykantov, Vladimir R

2006-01-01

Enzyme replacement therapy, a state-of-the-art treatment for many lysosomal storage disorders, relies on carbohydrate-mediated binding of recombinant enzymes to receptors that mediate lysosomal delivery via clathrin-dependent endocytosis. Suboptimal glycosylation of recombinant enzymes and deficiency of clathrin-mediated endocytosis in some lysosomal enzyme-deficient cells limit delivery and efficacy of enzyme replacement therapy for lysosomal disorders. We explored a novel delivery strategy utilizing nanocarriers targeted to a glycosylation- and clathrin-independent receptor, intercellular adhesion molecule (ICAM)-1, a glycoprotein expressed on diverse cell types, up-regulated and functionally involved in inflammation, a hallmark of many lysosomal disorders. We targeted recombinant human acid sphingomyelinase (ASM), deficient in types A and B Niemann-Pick disease, to ICAM-1 by loading this enzyme to nanocarriers coated with anti-ICAM. Anti-ICAM/ASM nanocarriers, but not control ASM or ASM nanocarriers, bound to ICAM-1-positive cells (activated endothelial cells and Niemann-Pick disease patient fibroblasts) via ICAM-1, in a glycosylation-independent manner. Anti-ICAM/ASM nanocarriers entered cells via CAM-mediated endocytosis, bypassing the clathrin-dependent pathway, and trafficked to lysosomes, where delivered ASM displayed stable activity and alleviated lysosomal lipid accumulation. Therefore, lysosomal enzyme targeting using nanocarriers targeted to ICAM-1 bypasses defunct pathways and may improve the efficacy of enzyme replacement therapy for lysosomal disorders, such as Niemann-Pick disease.

Distribution of events of positive selection and population differentiation in a metabolic pathway: the case of asparagine N-glycosylation

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Dall’Olio Giovanni

2012-06-01

Full Text Available Abstract Background Asparagine N-Glycosylation is one of the most important forms of protein post-translational modification in eukaryotes. This metabolic pathway can be subdivided into two parts: an upstream sub-pathway required for achieving proper folding for most of the proteins synthesized in the secretory pathway, and a downstream sub-pathway required to give variability to trans-membrane proteins, and involved in adaptation to the environment and innate immunity. Here we analyze the nucleotide variability of the genes of this pathway in human populations, identifying which genes show greater population differentiation and which genes show signatures of recent positive selection. We also compare how these signals are distributed between the upstream and the downstream parts of the pathway, with the aim of exploring how forces of population differentiation and positive selection vary among genes involved in the same metabolic pathway but subject to different functional constraints. Results Our results show that genes in the downstream part of the pathway are more likely to show a signature of population differentiation, while events of positive selection are equally distributed among the two parts of the pathway. Moreover, events of positive selection are frequent on genes that are known to be at bifurcation points, and that are identified as being in key position by a network-level analysis such as MGAT3 and GCS1. Conclusions These findings indicate that the upstream part of the Asparagine N-Glycosylation pathway has lower diversity among populations, while the downstream part is freer to tolerate diversity among populations. Moreover, the distribution of signatures of population differentiation and positive selection can change between parts of a pathway, especially between parts that are exposed to different functional constraints. Our results support the hypothesis that genes involved in constitutive processes can be expected to show

Glycosylation-mediated phenylpropanoid partitioning in Populus tremuloides cell cultures

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Babst Benjamin A

2009-12-01

Full Text Available Abstract Background Phenylpropanoid-derived phenolic glycosides (PGs and condensed tannins (CTs comprise large, multi-purpose non-structural carbon sinks in Populus. A negative correlation between PG and CT concentrations has been observed in several studies. However, the molecular mechanism underlying the relationship is not known. Results Populus cell cultures produce CTs but not PGs under normal conditions. Feeding salicyl alcohol resulted in accumulation of salicins, the simplest PG, in the cells, but not higher-order PGs. Salicin accrual reflected the stimulation of a glycosylation response which altered a number of metabolic activities. We utilized this suspension cell feeding system as a model for analyzing the possible role of glycosylation in regulating the metabolic competition between PG formation, CT synthesis and growth. Cells accumulated salicins in a dose-dependent manner following salicyl alcohol feeding. Higher feeding levels led to a decrease in cellular CT concentrations (at 5 or 10 mM, and a negative effect on cell growth (at 10 mM. The competition between salicin and CT formation was reciprocal, and depended on the metabolic status of the cells. We analyzed gene expression changes between controls and cells fed with 5 mM salicyl alcohol for 48 hr, a time point when salicin accumulation was near maximum and CT synthesis was reduced, with no effect on growth. Several stress-responsive genes were up-regulated, suggestive of a general stress response in the fed cells. Salicyl alcohol feeding also induced expression of genes associated with sucrose catabolism, glycolysis and the Krebs cycle. Transcript levels of phenylalanine ammonia lyase and most of the flavonoid pathway genes were reduced, consistent with down-regulated CT synthesis. Conclusions Exogenous salicyl alcohol was readily glycosylated in Populus cell cultures, a process that altered sugar utilization and phenolic partitioning in the cells. Using this system, we

Model-based investigation of intracellular processes determining antibody Fc-glycosylation under mild hypothermia.

Science.gov (United States)

Sou, Si Nga; Jedrzejewski, Philip M; Lee, Ken; Sellick, Christopher; Polizzi, Karen M; Kontoravdi, Cleo

2017-07-01

Despite the positive effects of mild hypothermic conditions on monoclonal antibody (mAb) productivity (q mAb ) during mammalian cell culture, the impact of reduced culture temperature on mAb Fc-glycosylation and the mechanism behind changes in the glycan composition are not fully established. The lack of knowledge about the regulation of dynamic intracellular processes under mild hypothermia restricts bioprocess optimization. To address this issue, a mathematical model that quantitatively describes Chinese hamster ovary (CHO) cell behavior and metabolism, mAb synthesis and mAb N-linked glycosylation profile before and after the induction of mild hypothermia is constructed. Results from this study show that the model is capable of representing experimental results well in all of the aspects mentioned above, including the N-linked glycosylation profile of mAb produced under mild hypothermia. Most importantly, comparison between model simulation results for different culture temperatures suggests the reduced rates of nucleotide sugar donor production and galactosyltransferase (GalT) expression to be critical contributing factors that determine the variation in Fc-glycan profiles between physiological and mild hypothermic conditions in stable CHO transfectants. This is then confirmed using experimental measurements of GalT expression levels, thereby closing the loop between the experimental and the computational system. The identification of bottlenecks within CHO cell metabolism under mild hypothermic conditions will aid bioprocess optimization, for example, by tailoring feeding strategies to improve NSD production, or manipulating the expression of specific glycosyltransferases through cell line engineering. Biotechnol. Bioeng. 2017;114: 1570-1582. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals Inc. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals Inc.

Oxytocin analogues with O-glycosylated serine and threonine in position 4

Czech Academy of Sciences Publication Activity Database

Marcinkowska, A.; Borovičková, Lenka; Slaninová, Jiřina; Grzonka, Z.

2007-01-01

Roč. 81, č. 7 (2007), s. 1335-1344 ISSN 0137- 5083 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z90210515 Keywords : oxytocin * glycosylated serin * glycosylated threonin * position 4 Subject RIV: CE - Biochemistry Impact factor: 0.483, year: 2007

Radiometallating antibodies and biologically active peptides

International Nuclear Information System (INIS)

Mercer-Smith, J.A.; Roberts, J.C.; Lewis, D.; Newmyer, S.L.; Schulte, L.D.; Burns, T.P.; Mixon, P.L.; Jeffery, A.L.; Schreyer, S.A.; Cole, D.A.; Figard, S.D.; Lennon, V.A.; Hayashi, M.; Lavallee, D.K.

1990-01-01

We have developed methods to radiolabel large molecules, using porphyrins as bifunctional chelating agents for radiometals. The porphyrins are substituted with an N-benzyl group to activate them for radiometallation under mild reaction conditions. Porphyrins that have on functional group for covalent attachment to other molecules cannot cause crosslinking. We have examined the labeling chemistry for antibodies, and we have also developed methods to label smaller biologically active molecules, such as autoantigenic peptides. The autoantigenic peptides, fragments of the acetylcholine receptor, are under investigation for myasthenia gravis research. The methods of covalent attachment of these bifunctional chelating agents to large molecules and the radiometallation chemistry will be discussed

Micropinocytic ingestion of glycosylated albumin by isolated microvessels: possible role in pathogenesis of diabetic microangiopathy.

OpenAIRE

Williams, S K; Devenny, J J; Bitensky, M W

1981-01-01

Microvessels isolated from rat epididymal fat exhibit differential vesicular ingestion rates for unmodified and non-enzymatically glycosylated rat albumin. While unmodified rat albumin is excluded from ingestion by endothelial micropinocytic vesicles, glycosylated albumin is avidly taken up by endocytosis. Interaction of albumin and glycosylated albumin with endothelium was studied with a double-label fluorescence assay of micropinocytosis. When glycosylated albumin was present at a concentra...

Amino acid and glucose metabolism in fed-batch CHO cell culture affects antibody production and glycosylation

DEFF Research Database (Denmark)

Fan, Yuzhou; Jimenez Del Val, Ioscani; Müller, Christian

2015-01-01

optimization, especially media optimization. Gaining knowledge on their interrelations could provide insight for obtaining higher immunoglobulin G (IgG) titer and better controlling glycosylationrelated product quality. In this work, different fed-batch processes with two chemically defined proprietary media......Fed-batch Chinese hamster ovary (CHO) cell culture is the most commonly used process for IgG production in the biopharmaceutical industry. Amino acid and glucose consumption, cell growth, metabolism, antibody titer, and N-glycosylation patterns are always the major concerns during upstream process...... and glutamine concentrations and uptake rates were positively correlated with intracellular UDP-Gal availability. All these findings are important for optimization of fed-batch culture for improving IgG production and directing glycosylation quality....

Nonenzymatic glycosylation of human hemoglobin at multiple sites

International Nuclear Information System (INIS)

Shapiro, R.; McManus, M.; Garrick, L.; McDonald, M.J.; Bunn, H.F.

1979-01-01

The most abundant minor hemoglobin component of human hemolysate is Hb A1c, which has glucose bound to the N-terminus of the beta chain by a ketoamine linkage. Hb A1c is formed slowly and continuously throughout the 120 day lifespan of the red cell. It can be synthesized in vitro by incubating purified hemoglobin with 14C-glucose. Other minor components, Hb A1a1 and Hb A1a2 are adducts of sugar phosphates at the N-terminus of the beta chain. Hb A1b contains an unidentified nonphosphorylated sugar at the beta N-terminus. In addition, a significant portion of the major hemoglobin component (Hb Ao) is also glycosylated by a glucose ketoamine linkage at other sites on the molecule, including the N-terminus of the alpha chain and the epsilon-amino group of several lysine residues on both the alpha and the beta chains. The results indicate that the interaction of glucose and hemoglobin is rather nonspecific and suggests that other proteins are modified in a similar fashion

Cooperative roles of glucose and asparagine-linked glycosylation in T-type calcium channel expression

Czech Academy of Sciences Publication Activity Database

Lazniewska, Joanna; Rzhepetskyy, Yuriy; Zhang, F. X.; Zamponi, G. W.; Weiss, Norbert

2016-01-01

Roč. 468, 11/12 (2016), s. 1837-1851 ISSN 0031-6768 R&D Projects: GA ČR GA15-13556S; GA MŠk 7AMB15FR015 Institutional support: RVO:61388963 Keywords : calcium channel * T-type channel * Ca(v)3.2 * glucose * N-glycosylation * trafficking Subject RIV: CE - Biochemistry Impact factor: 3.156, year: 2016

Dependency of the regio- and stereoselectivity of intramolecular, ring-closing glycosylations upon the ring size

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Patrick Claude

2011-12-01

Full Text Available Phenyl 3,4,6-tri-O-benzyl-2-O-(3-carboxypropionyl-1-thio-β-D-galactopyranoside (1 was condensed via its pentafluorophenyl ester 2 with 5-aminopentyl (4a, 4-aminobutyl (4b, 3-aminopropyl (4c and 2-aminoethyl 4,6-O-benzylidene-β-D-glucopyranoside (4d, prepared from the corresponding N-Cbz protected glucosides 3a–d, to give the corresponding 2-[3-(alkylcarbamoylpropionyl] tethered saccharides 5a–d. Intramolecular, ring closing glycosylation of the saccharides with NIS and TMSOTf afforded the tethered β(1→3 linked disaccharides 6a–c, the α(1→3 linked disaccharides 7a–d and the α(1→2 linked disaccharide 8d in ratios depending upon the ring size formed during glycosylation. No β(1→2 linked disaccharides were formed. Molecular modeling of saccharides 6–8 revealed that a strong aromatic stacking interaction between the aromatic parts of the benzyl and benzylidene protecting groups in the galactosyl and glucosyl moieties was mainly responsible for the observed regioselectivity and anomeric selectivity of the ring-closing glycosylation step.

N-linked glycans are required on epithelial Na+ channel subunits for maturation and surface expression.

Science.gov (United States)

Kashlan, Ossama B; Kinlough, Carol L; Myerburg, Michael M; Shi, Shujie; Chen, Jingxin; Blobner, Brandon M; Buck, Teresa M; Brodsky, Jeffrey L; Hughey, Rebecca P; Kleyman, Thomas R

2018-03-01

Epithelial Na + channel (ENaC) subunits undergo N-linked glycosylation in the endoplasmic reticulum where they assemble into an αβγ complex. Six, 13, and 5 consensus sites (Asn-X-Ser/Thr) for N-glycosylation reside in the extracellular domains of the mouse α-, β-, and γ-subunits, respectively. Because the importance of ENaC N-linked glycans has not been fully addressed, we examined the effect of preventing N-glycosylation of specific subunits on channel function, expression, maturation, and folding. Heterologous expression in Xenopus oocytes or Fischer rat thyroid cells with αβγ-ENaC lacking N-linked glycans on a single subunit reduced ENaC activity as well as the inhibitory response to extracellular Na + . The lack of N-linked glycans on the β-subunit also precluded channel activation by trypsin. However, channel activation by shear stress was N-linked glycan independent, regardless of which subunit was modified. We also discovered that the lack of N-linked glycans on any one subunit reduced the total and surface levels of cognate subunits. The lack of N-linked glycans on the β-subunit had the largest effect on total levels, with the lack of N-linked glycans on the γ- and α-subunits having intermediate and modest effects, respectively. Finally, channels with wild-type β-subunits were more sensitive to limited trypsin proteolysis than channels lacking N-linked glycans on the β-subunit. Our results indicate that N-linked glycans on each subunit are required for proper folding, maturation, surface expression, and function of the channel.

Analysis of expression and glycosylation of avian metapneumovirus attachment glycoprotein from recombinant baculoviruses.

Science.gov (United States)

Luo, Lizhong; Nishi, Krista; MacLeod, Erin; Sabara, Marta I; Li, Yan

2010-11-01

Recently, we reported the expression and glycosylation of avian metapneumovirus attachment glycoprotein (AMPV/C G protein) in eukaryotic cell lines by a transient-expression method. In the present study, we investigated the biosynthesis and O-linked glycosylation of the AMPV/C G protein in a baculovirus expression system. The results showed that the insect cell-produced G protein migrated more rapidly in SDS-PAGE as compared to LLC-MK2 cell-derived G proteins owing to glycosylation differences. The fully processed, mature form of G protein migrated between 78 and 86 kDa, which is smaller than the 110 kDa mature form of G expressed in LLC-MK2 cells. In addition, several immature G gene products migrating at 40-48 and 60-70 kDa were also detected by SDS-PAGE and represented glycosylated intermediates. The addition of the antibiotic tunicamycin, which blocks early steps of glycosylation, to insect cell culture resulted in the disappearance of two glycosylated forms of the G protein and identified a 38 kDa unglycosylated precursor. The maturation of the G protein was completely blocked by monensin, suggesting that the O-linked glycosylation of G initiated in the trans-Golgi compartment. The presence of O-linked sugars on the mature protein was further confirmed by lectin Arachis hypogaea binding assay. Furthermore, antigenic features of the G protein expressed in insect cells were evaluated by ELISA. Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.

The Length of N-Glycans of Recombinant H5N1 Hemagglutinin Influences the Oligomerization and Immunogenicity of Vaccine Antigen

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Edyta Kopera

2017-04-01

Full Text Available Hemagglutinin glycoprotein (HA is a principle influenza vaccine antigen. Recombinant HA-based vaccines become a potential alternative for traditional approach. Complexity and variation of HA N-glycosylation are considered as the important factors for the vaccine design. The number and location of glycan moieties in the HA molecule are also crucial. Therefore, we decided to study the effect of N-glycosylation pattern on the H5 antigen structure and its ability to induce immunological response. We also decided to change neither the number nor the position of the HA glycosylation sites but only the glycan length. Two variants of the H5 antigen with high mannose glycosylation (H5hm and with low-mannose glycosylation (H5Man5 were prepared utilizing different Pichia strains. Our structural studies demonstrated that only the highly glycosylated H5 antigen formed high molecular weight oligomers similar to viral particles. Further, the H5hm was much more immunogenic for mice than H5Man5. In summary, our results suggest that high mannose glycosylation of vaccine antigen is superior to the low glycosylation pattern. Our findings have strong implications for the recombinant HA-based influenza vaccine design.

Phytoremediation of Benzophenone and Bisphenol A by Glycosylation with Immobilized Plant Cells

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Kei Shimoda

2009-01-01

Full Text Available Benzophenone and bisphenol A are environmental pollutions, which have been listed among “chemicals suspected of having endocrine disrupting effects” by the World Wildlife Fund, the National Institute of Environmental Health Sciences in the USA and the Japanese Environment Agency. The cultured cells of Nicotiana tabacum glycosylated benzophenone to three glycosides, 4-O-β-D-glucopyranosylbenzophenone (9%, diphenylmethyl β-D-glucopyranoside (14%, and diphenylmethyl 6-O-(β-D-glucopyranosyl-β-D-glucopyranoside (12% after 48 h incubation. On the other hand, incubation of benzophenone with immobilized cells of N. tabacum in sodium alginate gel gave products in higher yields, i.e. the yields of 4-O-β-D-glucopyranosylbenzophenone, diphenylmethyl β-D-glucopyranoside, and diphenylmethyl 6-O-(β-D-glucopyranosyl-β-D-glucopyranoside were 15, 27, and 22%, respectively. Bisphenol A was converted into three glycosides, 2,2-bis(4-β-D-glucopyranosyloxyphenylpropane (16%, 2-(4-β-D-glucopyranosyloxy-3-hydroxyphenyl-2-(4-β-D-gluco- pyranosyloxyphenyl propane (8%, and 2-(3-β-D-glucopyranosyloxy-4-hydroxyphenyl-2-(4-β-D-glucopyranosyloxyphe nylpropane (5%. Also the use of immobilized N. tabacum cells improved the yield of products; the glycosylation of bisphenol A with immobilized N. tabacum gave 2,2-bis(4-β-D-glucopyranosyloxyphenylpropane (24%, 2-(4-β-D-gluco- pyranosyloxy-3-hydroxyphenyl-2-(4-β-D-glucopyranosyloxyphenyl propane (15%, and 2-(3-β-D-glucopyranosyloxy- 4-hydroxyphenyl-2-(4-β-D-glucopyranosyloxyphenylpropane (11%.

COMPARISON OF FRUCTOSAMINE AND GLYCOSYLATED HEMOGLOBIN IN A NON-INSULIN DEPENDENT DIABETIC POPULATION

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M. Amini

1999-08-01

Full Text Available In an attempt to determine the clinical value of frnctosamine assay for monitoring type II diabetic patients, correlation of frnctosamine with glycosylated hemoglobin was studied. 100 patients with type II diabetes mcllitus were compared with 100 normal subjects. Fasting blood glucose, glycosylated hemoglobin, albumin and frnctosamine were measured in alt subjects. In the diabetic patients, a significant correlation was observed between fasting blood glucose and glycosylated hemoglobin (r = 0.64, p < 0.01 and scrum frnctosamine (r = 0.7, P < 0.01. Tlicrc was also a significant correlation between glycosylated hemoglobin and scrum frtictosmine (r = .94, I'<0.01. Frnctosamine, assay can be used as an index of diabetes control.

Study on the relationship between blood levels of growth hormone, glycosylated hemoglobin and micro-vascular nephropathy in patients with diabetes

International Nuclear Information System (INIS)

Cai Facheng; Yao Yingfei; Zhang Jinchi

2005-01-01

Objective: To evaluate the relationship between blood levels of growth hormone, glycosylated hemoglobin and micro-vascular nephropathy in patients with diabetes. Methods: Blood growth hormone and β 2 -m levels were determined with RIA and GH2 bA 1C , blood glucose were determined with biochemical method in 41 diabetic patients and 32 controls. Results: The blood levels of growth hormone, glycosylated hemoglobin, β 2 -microglobulin and fasting blood glucose in the patients with diabetes well controlled (n=22) were significantly higher than those in controls and levels in patients with diabetes poorly controlled (n=19) were again significantly higher than those in patients with diabetes well controlled (P 2 -microglobulin and fasting blood glucose is very important for early detection of diabetic nephropathy. (authors)

Cell culture media supplementation of uncommonly used sugars sucrose and tagatose for the targeted shifting of protein glycosylation profiles of recombinant protein therapeutics.

Science.gov (United States)

Hossler, Patrick; McDermott, Sean; Racicot, Christopher; Chumsae, Christopher; Raharimampionona, Haly; Zhou, Yu; Ouellette, David; Matuck, Joseph; Correia, Ivan; Fann, John; Li, Jianmin

2014-01-01

Protein glycosylation is an important post-translational modification toward the structure and function of recombinant therapeutics. The addition of oligosaccharides to recombinant proteins has been shown to greatly influence the overall physiochemical attributes of many proteins. It is for this reason that protein glycosylation is monitored by the developer of a recombinant protein therapeutic, and why protein glycosylation is typically considered a critical quality attribute. In this work, we highlight a systematic study toward the supplementation of sucrose and tagatose into cell culture media for the targeted modulation of protein glycosylation profiles on recombinant proteins. Both sugars were found to affect oligosaccharide maturation resulting in an increase in the percentage of high mannose N-glycan species, as well as a concomitant reduction in fucosylation. The latter effect was demonstrated to increase antibody-dependent cell-mediated cytotoxicity for a recombinant antibody. These aforementioned results were found to be reproducible at different scales, and across different Chinese hamster ovary cell lines. Through the selective supplementation of these described sugars, the targeted modulation of protein glycosylation profiles is demonstrated, as well as yet another tool in the cell culture toolbox for ensuring product comparability. © 2014 American Institute of Chemical Engineers.

A novel Meloidogyne graminicola effector, MgGPP, is secreted into host cells and undergoes glycosylation in concert with proteolysis to suppress plant defenses and promote parasitism.

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Jiansong Chen

2017-04-01

Full Text Available Plant pathogen effectors can recruit the host post-translational machinery to mediate their post-translational modification (PTM and regulate their activity to facilitate parasitism, but few studies have focused on this phenomenon in the field of plant-parasitic nematodes. In this study, we show that the plant-parasitic nematode Meloidogyne graminicola has evolved a novel effector, MgGPP, that is exclusively expressed within the nematode subventral esophageal gland cells and up-regulated in the early parasitic stage of M. graminicola. The effector MgGPP plays a role in nematode parasitism. Transgenic rice lines expressing MgGPP become significantly more susceptible to M. graminicola infection than wild-type control plants, and conversely, in planta, the silencing of MgGPP through RNAi technology substantially increases the resistance of rice to M. graminicola. Significantly, we show that MgGPP is secreted into host plants and targeted to the ER, where the N-glycosylation and C-terminal proteolysis of MgGPP occur. C-terminal proteolysis promotes MgGPP to leave the ER, after which it is transported to the nucleus. In addition, N-glycosylation of MgGPP is required for suppressing the host response. The research data provide an intriguing example of in planta glycosylation in concert with proteolysis of a pathogen effector, which depict a novel mechanism by which parasitic nematodes could subjugate plant immunity and promote parasitism and may present a promising target for developing new strategies against nematode infections.

Scanning the available Dictyostelium discoideum proteome for O-linked GlcNAc glycosylation sitesusing neural networks

DEFF Research Database (Denmark)

Gupta, Ramneek; Jung, Eva; Gooley, Andrew A

1999-01-01

Dictyostelium discoideum has been suggested as a eukaryotic model organism for glycobiology studies. Presently, the characteristics of acceptor sites for the N-acetylglucosaminyl-transferases in Dictyostelium discoideum, which link GlcNAc in an alpha linkage to hydroxyl residues, are largely...... unknown. This motivates the development of a species specific method for prediction of O-linked GlcNAc glycosylation sites in secreted and membrane proteins of D. discoideum. The method presented here employs a jury of artificial neural networks. These networks were trained to recognize the sequence...... context and protein surface accessibility in 39 experimentally determined O-alpha-GlcNAc sites found in D. discoideum glycoproteins expressed in vivo. Cross-validation of the data revealed a correlation in which 97% of the glycosylated and nonglycosylated sites were correctly identified. Based...

Reduction of N-linked xylose and fucose by expression of rat beta1,4-N-acetylglucosaminyltransferase III in tobacco BY-2 cells depends on Golgi enzyme localization domain and genetic elements used for expression.

Science.gov (United States)

Karg, Saskia R; Frey, Alexander D; Kallio, Pauli T

2010-03-01

Plant-specific N-glycosylation, such as the introduction of core alpha1,3-fucose and beta1,2-xylose residues, is a major obstacle to the utilization of plant cell- or plant-derived recombinant therapeutic proteins. The beta1,4-N-acetylglucosaminyltransferase III (GnTIII) introduces a bisecting GlcNAc residue into N-glycans, which exerts a high level of substrate mediated control over subsequent modifications, for example inhibiting mammalian core fucosylation. Based on similar findings in plants, we used Nicotianatabacum BY-2 cells to study the effects of localization and expression levels of GnTIII in the remodeling of the plant N-glycosylation pathway. The N-glycans produced by the cells expressing GnTIII were partially bisected and practically devoid of the paucimannosidic type which is typical for N-glycans produced by wildtype BY-2 suspension cultured cells. The proportion of human-compatible N-glycans devoid of fucose and xylose could be increased from an average of 4% on secreted protein from wildtype cells to as high as 59% in cells expressing chimeric GnTIII, named GnTIII(A.th.) replacing its native localization domain with the cytoplasmic tail, transmembrane, and stem region of Arabidopsis thaliana mannosidase II. The changes in N-glycosylation observed were dependent on the catalytic activity of GnTIII, as the expression of catalytically inactive GnTIII mutants did not show a significant effect on N-glycosylation. Copyright 2010 Elsevier B.V. All rights reserved.

Identification of N-glycosylation in prolyl endoprotease from Aspergillus niger and evaluation of the enzyme for its possible application in proteomics.

Science.gov (United States)

Sebela, Marek; Rehulka, Pavel; Kábrt, Jaromír; Rehulková, Helena; Ozdian, Tomás; Raus, Martin; Franc, Vojtech; Chmelík, Josef

2009-11-01

An acidic prolyl endoprotease from Aspergillus niger was isolated from the commercial product Brewers Clarex to evaluate its possible application in proteomics. The chromatographic purification yielded a single protein band in sodium dodecyl sulfate polyacrylamide gel electrophoresis providing an apparent molecular mass of 63 kDa and a broad peak (m/z 58,061) in linear matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) indicating the glycoprotein nature of the enzyme. Indeed, a colorimetric assessment with phenol and sulfuric acid showed the presence of neutral sugars (9% of weight). The subsequent treatment with N-glycosidase F released a variety of high-mannose type N-glycans, which were successfully detected using MALDI-TOF MS. MALDI-TOF/TOF tandem MS analysis of glycopeptides from a tryptic digest of prolyl endoprotease unraveled the identity of the N-glycosylation site in the primary structure. The data obtained also show that the enzyme is present in its processed form, i.e. without putative signal and propeptide parts. Spectrophotometric measurements demonstrated optimal activity at pH 4.0-4.5 and also high thermostability for the cleavage at the C-terminal part of proline residues. In-solution digestion of standard proteins (12-200 kDa) allowed to evaluate the cleavage specificity. The enzyme acts upon proline and alanine residues, but there is an additional minor cleavage at some other residues like Gly, Leu, Arg, Ser and Tyr. The digestion of a honeybee peptide comprising six proline residues (apidaecin 1A) led to the detection of specific peptides terminated by proline as it was confirmed by MALDI postsource decay analysis. Copyright 2009 John Wiley & Sons, Ltd.

Morphology, histochemistry and glycosylation of the placenta and associated tissues in the European hedgehog (Erinaceus europaeus)

DEFF Research Database (Denmark)

Jones, Carolyn J P; Carter, A M; Allen, W R

2016-01-01

glycosylated. Yolk sac inner and outer endoderm expressed similar glycans except for N-acetylgalactosamine residues in endodermal acini. DISCUSSION: New features of near-term hedgehog placenta and associated tissues are presented, including their glycosylation, and novel yolk sac acinar structures......INTRODUCTION: There are few descriptions of the placenta and associated tissues of the European hedgehog (Erinaceus europaeus) and here we present findings on a near-term pregnant specimen. METHODS: Tissues were examined grossly and then formalin fixed and wax-embedded for histology...... and immunocytochemistry (cytokeratin) and resin embedded for lectin histochemistry. RESULTS: Each of four well-developed and near term hoglets displayed a discoid, haemochorial placenta with typical labyrinth and spongy zones. In addition there was a paraplacenta incorporating Reichert's membrane and a largely detached...

Production of Active Nonglycosylated Recombinant B-Chain of Type-2 Ribosome-Inactivating Protein from Viscum articulatum and Its Biological Effects on Peripheral Blood Mononuclear Cells

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Tzu-Li Lu

2011-01-01

Full Text Available Type-2 ribosome-inactivating proteins, composed of a toxic A-chain and lectin-like B-chain, display various biological functions, including cytotoxicity and immunomodulation. We here cloned the lectin-like B-chain encoding fragment of a newly identified type-2 RIP gene, articulatin gene, from Viscum articulatum, into a bacterial expression vector to obtain nonglycosylated recombinant protein expressed in inclusion bodies. After purification and protein refolding, soluble refolded recombinant articulatin B-chain (rATB showed lectin activity specific toward galactoside moiety and was stably maintained while stored in low ionic strength solution. Despite lacking glycosylation, rATB actively bound leukocytes with preferential binding to monocytes and in vitro stimulated PBMCs to release cytokines without obvious cytotoxicity. These results implicated such a B-chain fragment as a potential immunomodulator.

Posttranslational modifications of proopiomelanocortin in vertebrates and their biological significance

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Akiyoshi eTakahashi

2013-10-01

Full Text Available Proopiomelanocortin (POMC is the precursor of several peptide hormones generated in the pituitary gland. After biosynthesis, POMC undergoes several posttranslational modifications, including proteolytic cleavage, acetylation, amidation, phosphorylation, glycosylation, and disulfide linkage formation, which generate mature POMC-derived peptides. Therefore, POMC is a useful model for the investigation of posttranslational modifications. These processes have been extensively investigated in mammals, primarily in rodents. In addition, over the last decade, much information has been obtained about the posttranslational processing of POMC in non-mammalian animals such as fish, amphibians, reptiles, and birds through sequencing and peptide identification by mass spectrometry. One POMC modification, acetylation, is known to modulate the biological activities of POMC-derived alpha-melanocyte-stimulating hormone (alpha-MSH having an acetyl group at N-terminal through potentiation or inhibition. This bidirectional regulation depends on its intrinsic roles in the tissue or cell; for example, alpha-MSH, as well as desacety-alpha-MSH, stimulates pigment dispersion in the xanthophores of a flounder. In contrast, alpha-MSH does not stimulate pigment dispersion in the melanophores of the same species, whereas desacetyl-alpha-MSH does. Regulation of pigment-dispersing activities may be associated with the subtle balance in the expression of receptor genes. In this review, we consider the posttranslational modifications of POMC in vertebrates from an evolutionary aspect, with a focus on the relationship between acetylation and the biological activities of alpha-MSH as an important consequence of posttranslational modification.

Glycosylation is essential for translocation of carp retinol-binding protein across the endoplasmic reticulum membrane

International Nuclear Information System (INIS)

Devirgiliis, Chiara; Gaetani, Sancia; Apreda, Marianna; Bellovino, Diana

2005-01-01

Retinoid transport is well characterized in many vertebrates, while it is still largely unexplored in fish. To study the transport and utilization of vitamin A in these organisms, we have isolated from a carp liver cDNA library retinol-binding protein, its plasma carrier. The primary structure of carp retinol-binding protein is very conserved, but presents unique features compared to those of the correspondent proteins isolated and characterized so far in other species: it has an uncleavable signal peptide and two N-glycosylation sites in the NH 2 -terminal region of the protein that are glycosylated in vivo. In this paper, we have investigated the function of the carbohydrate chains, by constructing three mutants deprived of the first, the second or both carbohydrates. The results of transient transfection of wild type and mutant retinol-binding protein in Cos cells followed by Western blotting and immunofluorescence analysis have shown that the absence of both carbohydrate moieties blocks secretion, while the presence of one carbohydrate group leads to an inefficient secretion. Experiments of carp RBP mRNA in vitro translation in a reticulocyte cell-free system in the presence of microsomes have demonstrated that N-glycosylation is necessary for efficient translocation across the endoplasmic reticulum membranes. Moreover, when Cos cells were transiently transfected with wild type and mutant retinol-binding protein (aa 1-67)-green fluorescent protein fusion constructs and semi-permeabilized with streptolysin O, immunofluorescence analysis with anti-green fluorescent protein antibody revealed that the double mutant is exposed to the cytosol, thus confirming the importance of glycan moieties in the translocation process

Effect of Ozonation and Biological Activated Carbon Treatment of Wastewater Effluents on Formation of N-nitrosamines and Halogenated Disinfection Byproducts.

Science.gov (United States)

Chuang, Yi-Hsueh; Mitch, William A

2017-02-21

Ozonation followed by biological activated carbon (O 3 /BAC) is being considered as a key component of reverse osmosis-free advanced treatment trains for potable wastewater reuse. Using a laboratory-scale O 3 /BAC system treating two nitrified wastewater effluents, this study characterized the effect of different ozone dosages (0-1.0 mg O 3 /mg dissolved organic carbon) and BAC empty bed contact times (EBCT; 15-60 min) on the formation after chlorination or chloramination of 35 regulated and unregulated halogenated disinfection byproducts (DBPs), 8 N-nitrosamines, and bromate. DBP concentrations were remarkably similar between the two wastewaters across O 3 /BAC conditions. Ozonation increased bromate, TCNM, and N-nitrosodimethylamine, but ozonation was less significant for other DBPs. DBP formation generally decreased significantly with BAC treatment at 15 min EBCT, but little further reduction was observed at higher EBCT where low dissolved oxygen concentrations may have limited biological activity. The O 3 /BAC-treated wastewaters met regulatory levels for trihalomethanes (THMs), haloacetic acids (HAAs), and bromate, although N-nitrosodimethylamine exceeded the California Notification Level in one case. Regulated THMs and HAAs dominated by mass. When DBP concentrations were weighted by measures of their toxic potencies, unregulated haloacetonitriles, haloacetaldehydes, and haloacetamides dominated. Assuming toxicity is additive, the calculated DBP-associated toxicity of the O 3 /BAC-treated chloraminated effluents were comparable or slightly higher than those calculated in a recent evaluation of Full Advanced Treatment trains incorporating reverse osmosis.

Synthesis and biological activity of a new class of insecticides: the N-(5-aryl-1,3,4-thiadiazol-2-yl)amides.

Science.gov (United States)

Eckelbarger, Joseph D; Parker, Marshall H; Yap, Maurice Ch; Buysse, Ann M; Babcock, Jonathan M; Hunter, Ricky; Adelfinskaya, Yelena; Samaritoni, Jack G; Garizi, Negar; Trullinger, Tony K

2017-04-01

Optimization studies on a high-throughput screening (HTS) hit led to the discovery of a series of N-(6-arylpyridazin-3-yl)amides with insecticidal activity. It was hypothesized that the isosteric replacement of the pyridazine ring with a 1,3,4-thiadiazole ring could lead to more potent biological activity and/or a broader sap-feeding pest spectrum. The resulting N-(5-aryl-1,3,4-thiadiazol-2-yl)amides were explored as a new class of insecticides. Several methods for 2-amino-1,3,4-thiadiazole synthesis were used for the preparation of key synthetic intermediates. Subsequent coupling to variously substituted carboxylic acid building blocks furnished the final targets, which were tested for insecticidal activity against susceptible strains of Aphis gossypii (Glover) (cotton aphid), Myzus persicae (Sulzer) (green peach aphid) and Bemisia tabaci (Gennadius) (sweetpotato whitefly). Structure-activity relationship (SAR) studies on both the amide tail and the aryl A-ring of novel N-(5-aryl-1,3,4-thiadiazol-2-yl)amides led to a new class of insecticidal molecules active against sap-feeding insect pests. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

O-GLYCOBASE version 4.0: a revised database of O-glycosylated proteins

DEFF Research Database (Denmark)

Gupta, Ramneek; Birch, Hanne; Rapacki, Krzysztof

1999-01-01

O-GLYCBASE is a database of glycoproteins with O-linked glycosylation sites. Entries with at least one experimentally verified O-glycosylation site have been complied from protein sequence databases and literature. Each entry contains information about the glycan involved, the species, sequence, ...

In Vitro Membrane Permeation Studies and in Vivo Antinociception of Glycosylated Dmt(1)-DALDA Analogues

DEFF Research Database (Denmark)

Ballet, Steven; Betti, Cecilia; Novoa, Alexandre

2014-01-01

In this study the μ opioid receptor (MOR) ligands DALDA (Tyr-d-Arg-Phe-Lys-NH2) and Dmt(1)-DALDA (Dmt-d-Arg-Phe-Lys-NH2, Dmt = 2',6'-dimethyltyrosine) were glycosylated at the N- or C-terminus. Subsequently, the modified peptides were subjected to in vitro and in vivo evaluation. In contrast to t...

Influence of intramolecular hydrogen bonds on regioselectivity of glycosylation. Synthesis of lupane-type saponins bearing the OSW-1 saponin disaccharide unit and its isomers

Czech Academy of Sciences Publication Activity Database

Kuczynska, K.; Cmoch, P.; Rárová, L.; Oklešťková, Jana; Korda, A.; Pakulski, Z.; Strnad, Miroslav

2016-01-01

Roč. 423, MAR 24 (2016), s. 49-69 ISSN 0008-6215 R&D Projects: GA MŠk(CZ) LO1204; GA ČR GA14-19590S Institutional support: RVO:61389030 Keywords : OSW-1 disaccharide * Glycosylation * Lupane saponins Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.096, year: 2016

SEM visualization of glycosylated surface molecules using lectin-coated microspheres

Science.gov (United States)

Duke, J.; Janer, L.; Campbell, M.

1985-01-01

There are several techniques currently used to localize glycosylated surface molecules by scanning electron microscopy (Grinnell, 1980; Molday, 1976; Linthicum and Sell, 1975; Nicolson, 1974; Lo Buglio, et al, 1972). A simple and rapid method, using a modification of Grinnell's technique is reported here. Essentially, microspheres coated with Concavalin A are used to bind to glycosylated regions of the palatal shelf epithelium and are visualized in the scanning electron microscope (SEM).

Sterol-recognition ability and membrane-disrupting activity of Ornithogalum saponin OSW-1 and usual 3-O-glycosyl saponins.

Science.gov (United States)

Malabed, Raymond; Hanashima, Shinya; Murata, Michio; Sakurai, Kaori

2017-12-01

OSW-1 is a structurally unique steroidal saponin isolated from the bulbs of Ornithogalum saundersiae, and has exhibited highly potent and selective cytotoxicity in tumor cell lines. This study aimed to investigate the molecular mechanism for the membrane-permeabilizing activity of OSW-1 in comparison with those of other saponins by using various spectroscopic approaches. The membrane effects and hemolytic activity of OSW-1 were markedly enhanced in the presence of membrane cholesterol. Binding affinity measurements using fluorescent cholestatrienol and solid-state NMR spectroscopy of a 3-d-cholesterol probe suggested that OSW-1 interacts with membrane cholesterol without forming large aggregates while 3-O-glycosyl saponin, digitonin, forms cholesterol-containing aggregates. The results suggest that OSW-1/cholesterol interaction is likely to cause membrane permeabilization and pore formation without destroying the whole membrane integrity, which could partly be responsible for its highly potent cell toxicity. Copyright © 2017 Elsevier B.V. All rights reserved.

Structural and Functional Consequences of Increased Tubulin Glycosylation in Diabetes Mellitus

Science.gov (United States)

Williams, Stuart K.; Howarth, Nancy L.; Devenny, James J.; Bitensky, Mark W.

1982-11-01

The extent of in vitro nonenzymatic glycosylation of purified rat brain tubulin was dependent on time and glucose concentration. Tubulin glycosylation profoundly inhibited GTP-dependent tubulin polymerization. Electron microscopy and NaDodSO4/polyacrylamide gel electrophoresis showed that glycosylated tubulin forms high molecular weight amorphous aggregates that are not disrupted by detergents or reducing agents. The amount of covalently bound NaB3H4-reducible sugars in tubulin recovered from brain of streptozotocin-induced diabetic rats was dramatically increased as compared with tubulin recovered from normal rat brain. Moreover, tubulin recovered from diabetic rat brain exhibited less GTP-induced polymerization than tubulin from nondiabetic controls. The possible implications of these data for diabetic neuropathy are discussed.

Interfacing nanostructures to biological cells

Science.gov (United States)

Chen, Xing; Bertozzi, Carolyn R.; Zettl, Alexander K.

2012-09-04

Disclosed herein are methods and materials by which nanostructures such as carbon nanotubes, nanorods, etc. are bound to lectins and/or polysaccharides and prepared for administration to cells. Also disclosed are complexes comprising glycosylated nanostructures, which bind selectively to cells expressing glycosylated surface molecules recognized by the lectin. Exemplified is a complex comprising a carbon nanotube functionalized with a lipid-like alkane, linked to a polymer bearing repeated .alpha.-N-acetylgalactosamine sugar groups. This complex is shown to selectively adhere to the surface of living cells, without toxicity. In the exemplified embodiment, adherence is mediated by a multivalent lectin, which binds both to the cells and the .alpha.-N-acetylgalactosamine groups on the nanostructure.

Publishing activities improves undergraduate biology education.

Science.gov (United States)

Smith, Michelle K

2018-06-01

To improve undergraduate biology education, there is an urgent need for biology instructors to publish their innovative active-learning instructional materials in peer-reviewed journals. To do this, instructors can measure student knowledge about a variety of biology concepts, iteratively design activities, explore student learning outcomes and publish the results. Creating a set of well-vetted activities, searchable through a journal interface, saves other instructors time and encourages the use of active-learning instructional practices. For authors, these publications offer new opportunities to collaborate and can provide evidence of a commitment to using active-learning instructional techniques in the classroom.

O-GLYCBASE version 3.0: a revised database of O-glycosylated proteins

DEFF Research Database (Denmark)

Hansen, Jan; Lund, Ole; Nilsson, Jette

1998-01-01

O-GLYCBASE is a revised database of information on glycoproteins and their O-linked glycosylation sites. Entries are compiled and revised from the literature, and from the sequence databases. Entries include informations about species, sequence, glycosylation sites and glycan type and is fully cr...

Patterns of glycemic control using glycosylated hemoglobin in diabetics.

Science.gov (United States)

Kahlon, Arunpreet Singh; Pathak, Rambha

2011-07-01

Till now estimation of blood glucose is the highly effective method for diagnosing diabetes mellitus but it provides a short-term picture of control. More evidence is required to prove that plasma glucose and glycosylated hemoglobin levels together gives a better estimate of glycemic control and compliance with treatment. Indian diabetes risk score (IDRS) is a simplified screening tool for identifying undiagnosed diabetic subjects, requires minimum time, and effort and can help to considerably reduce the costs of screening. To study patterns of glycemic control using glycosylated hemoglobin in diabetic patients. To find out correlation between levels of plasma glucose and glycosylated hemoglobin in diabetics and to calculate IDRS of the study population. A cross sectional study was conducted among 300 known diabetic patients attending outpatient department of a rural medical college in Haryana, India. Following standard procedures and protocols FPG and glycosylated hemoglobin were measured to find out a pattern of glycemic control in them after taking their written and informed consent. A correlation between the levels of glycosylated hemoglobin and fasting blood glucose was also calculated. These patients were made to fill a performa and their demographic and clinical risk factors were noted and based on this, their IDRS was calculated. This was done to validate the IDRS in Indian rural population. Fifty-two percent of the population had fasting plasma glucose level between 125-150 mg/dl, 21% had this level between 151-175 mg/dl. Thirteen percent of the study subjects had HbA1C between 6.5-7.5, more than half (57.3%) had this value between 7.5-8.5, 12% and 18% had values between 8.5-9.5 and 9.5-10.5, respectively. Twelve percent of the participants had HbA1C level higher than 10.5. Correlation of fasting plasma glucose level and HbA1C was also studied and found that correlation coefficient came out to be .311. This correlation was found to be statistically

Condensation reactions catalyzed by α-N-acetylgalactosaminidase from Aspergillus niger yielding a-N-acetylgalactosaminides

Czech Academy of Sciences Publication Activity Database

Weignerová, Lenka; Pelantová, Helena; Manglová, Dana; Michálková, Klára; Křen, Vladimír

2010-01-01

Roč. 28, č. 2 (2010), s. 150-155 ISSN 1024-2422 R&D Projects: GA MŠk OC 136 Keywords : alpha-N-Acetylgalactosaminidase * amino acid glycosylation * enzymatic glycosylation Subject RIV: EE - Microbiology, Virology Impact factor: 1.275, year: 2010

O-Glycosylation Modulates Proprotein Convertase Activation of Angiopoietin-like Protein 3: POSSIBLE ROLE OF POLYPEPTIDE GalNAc-TRANSFERASE-2 IN REGULATION OF CONCENTRATIONS OF PLASMA LIPIDS

DEFF Research Database (Denmark)

Schjoldager, Katrine Ter-Borch Gram; Vester-Christensen, Malene B; Bennett, Eric Paul

2010-01-01

immediately C-terminal (TT(226)). We developed an in vivo model system in CHO ldlD cells that was used to show that O-glycosylation in the processing site blocked processing of ANGPTL3. Genome-wide SNP association studies have identified the polypeptide GalNAc-transferase gene, GALNT2, as a candidate gene...... for low HDL and high triglyceride blood levels. We hypothesized that the GalNAc-T2 transferase performed critical O-glycosylation of proteins involved in lipid metabolism. Screening of a panel of proteins known to affect lipid metabolism for potential sites glycosylated by GalNAc-T2 led to identification...

N-Glycosylation of an IgG antibody secreted by Nicotiana tabacum BY-2 cells can be modulated through co-expression of human β-1,4-galactosyltransferase.

Science.gov (United States)

Navarre, Catherine; Smargiasso, Nicolas; Duvivier, Laurent; Nader, Joseph; Far, Johann; De Pauw, Edwin; Boutry, Marc

2017-06-01

Nicotiana tabacum BY-2 suspension cells have several advantages that make them suitable for the production of full-size monoclonal antibodies which can be purified directly from the culture medium. Carbohydrate characterization of an antibody (Lo-BM2) expressed in N. tabacum BY-2 cells showed that the purified Lo-BM2 displays N-glycan homogeneity with a high proportion (>70%) of the complex GnGnXF glycoform. The stable co-expression of a human β-1,4-galactosyltransferase targeted to different Golgi sub-compartments altered Lo-BM2N-glycosylation and resulted in the production of an antibody that exhibited either hybrid structures containing a low abundance of the plant epitopes (α-1,3-fucose and β-1,2-xylose), or a large amount of galactose-extended N-glycan structures. These results demonstrate the suitability of stable N-glycoengineered N. tabacum BY-2 cell lines for the production of human-like antibodies.

Soybean seed treatment with nickel improves biological nitrogen fixation and urease activity

Directory of Open Access Journals (Sweden)

José eLavres Junior

2016-05-01

Full Text Available Nickel (Ni is an essential micronutrient required for plants’ metabolism due to its role as a structural component of urease and hydrogenase, which in turn perform nitrogen (N metabolism in many legume species. Seed treatment with cobalt, molybdenum and Bradyrhizobium strains has been widely practiced to improve crops. Additionally, seed treatment together with Ni fertilization of soybean might improve the efficiency of biological nitrogen fixation (BNF, boosting grain dry matter yield and N content. The objective of this study was to evaluate the effect of soybean seed treatment with Ni rates (0, 45, 90,135, 180, 360 and 540 mg kg-1 on biological nitrogen fixation (BNF, directly by the 15N natural abundance method (δ15N‰ and by measurement of urease [E.C. 3.5.1.5] activity, as well as indirectly by nitrogenase (N-ase activity [E.C. 1.18.6.1]. Soybean plants (cultivar BMX Potência RR were grown in a sandy soil up to the R7 developmental stage (grain maturity, at which point the nutrient content in the leaves, chlorophyll content, urease and N-ase activities, Ni and N content in the grains, nodulation (at R1 - flowering stage, as well as the contribution of biological nitrogen fixation (δ15N ‰, were evaluated. The proportion of N derived from N2 fixation varied from 77 to 99% using the natural 15N abundance method and non-nodulating Panicum miliaceum and Phalaris canariensis as references. A Ni rate of 45 mg kg-1 increased BNF by 12% compared to the control. The increased N uptake in the grains was closely correlated with chlorophyll content in the leaves, urease and N-ase activities, as well as with nodulation. Grain dry matter yield and aerial part dry matter yield increased, respectively, by 84% and 51% in relation to the control plants at 45 mg kg-1 Ni via seed treatment. Despite, Ni concentration was increased with Ni-seed treatment, Ni rates higher than 135 mg kg-1 promoted negative effects on plant growth and yield. In these

Fasting serum glucose and glycosylated hemoglobin level in obesity.

Science.gov (United States)

Das, R K; Nessa, A; Hossain, M A; Siddiqui, N I; Hussain, M A

2014-04-01

Obesity is a condition in which the body fat stores are increased to an extent which impairs health and leads to serious health consequences. The amount of body fat is difficult to measure directly, and is usually determined from an indirect measure - the body mass index (BMI). Increased BMI in obese persons is directly associated with an increase in metabolic disease, such as type 2 diabetes mellitus. This Analytical cross sectional study was undertaken to assess the relation between obesity and glycemic control of body by measuring fasting serum glucose and glycosylated hemoglobin. This study was carried out in the Department of Physiology, Mymensingh Medical College, Mymensingh from 1st July 2011 to 30th June 2012 on 120 equally divided male and female persons within the age range of 25 to 55 years. Age more than 55 years and less than 25 years and diagnosed case of Hypothyroidism, Cushing's syndrome, polycystic ovary, Antipsychotic drug user and regular steroid users were excluded. Non probability purposive type of sampling technique was used for selecting the study subjects. Measurement of body mass index was done as per procedure. Fasting serum glucose was estimated by glucose oxidase method and Glycosylated hemoglobin by Boronate Affinity method. Statistical analysis was done by SPSS (version 17.0). Data were expressed as Mean±SE and statistical significance of difference among the groups were calculated by unpaired student's 't' test and Pearson's correlation coefficient tests were done as applicable. The Mean±SE of fasting serum glucose was significant at 1% level (P value obese group of BMI. There was no significant difference of glycosylated hemoglobin level between control and study groups. But there was positive correlation within each group. Fasting serum glucose also showed a bit stronger positive correlation with BMI. Both obese male and female persons showed higher levels of fasting serum glucose and glycosylated hemoglobin. The observed positive

The sweet side of a long pentraxin: how glycosylation affects PTX3 functions in innate immunity and inflammation

Directory of Open Access Journals (Sweden)

Antonio eInforzato

2013-01-01

Full Text Available Innate immunity represents the first line of defence against pathogens and plays key roles in activation and orientation of the adaptive immune response. The innate immune system comprises both a cellular and a humoral arm. Components of the humoral arm include soluble pattern recognition molecules (PRMs that recognise pathogen associated molecular patterns (PAMPs and initiate the immune response in coordination with the cellular arm, therefore acting as functional ancestors of antibodies. The long pentraxin PTX3 is a prototypic soluble PRM that is produced at sites of infection and inflammation by both somatic and immune cells. Gene targeting of this evolutionarily conserved protein has revealed a non-redundant role in resistance to selected pathogens. Moreover, PTX3 exerts important functions at the crossroad between innate immunity, inflammation and female fertility. The human PTX3 protein contains a single N-glycosylation site that is fully occupied by complex type oligosaccharides, mainly fucosylated and sialylated biantennary glycans. Glycosylation has been implicated in a number of PTX3 activities, including neutralization of influenza viruses, modulation of the complement system, and attenuation of leukocyte recruitment. Therefore, this post translational modification might act as a fine tuner of PTX3 functions in native immunity and inflammation.Here we review the studies on PTX3, with emphasis on the glycan-dependent mechanisms underlying pathogen recognition and crosstalk with other components of the innate immune system.

Intestinal epithelial cell surface glycosylation in mice. I. Effect of high-protein diet.

Science.gov (United States)

Gupta, R; Jaswal, V M; Meenu Mahmood, A

1992-01-01

The effects of variation in dietary protein content have been investigated on brush border glycosylation and enzyme activities in mice small intestine. The comparison of different parameters was made between the mice fed 30% (high protein, HP) and 18% protein (pair-fed, PF, and ad libitum-fed) for 21 days. The activities of brush border sucrase, lactase, p-nitrophenyl (PNP)-beta-D-glucosidase and PNP-beta-D-galactosidase were reduced in the HP diet-fed mice compared to PF and ad libitum-fed controls. Alkaline phosphatase and leucine amino-peptidase activities were significantly enhanced while gamma-glutamyl transpeptidase activity was unaltered under these conditions. Total hexoses and sialic acid content in the brush borders were reduced significantly in the test group compared to the controls while hexosamine and fucose contents remained essentially similar in different groups. The results on the binding of wheat germ agglutinin and Ulex europaeus agglutininI to microvillus membranes corroborated the chemical analysis data on sialic acid and fucose contents of the membranes. Peanut agglutinin binding was enhanced in mice from the HP group. Incorporation of (14C)-mannose into membranes was significantly less in HP diet-fed mice. These results indicate that the feeding of HP diet to mice brings about marked alterations in small intestinal epithelial cell surface glycosylation and enzyme functions.

Preparation and characterization of new biologically active polyurethane foams.

Science.gov (United States)

Savelyev, Yuri; Veselov, Vitali; Markovskaya, Ludmila; Savelyeva, Olga; Akhranovich, Elena; Galatenko, Natalya; Robota, Ludmila; Travinskaya, Tamara

2014-12-01

Biologically active polyurethane foams are the fast-developed alternative to many applications of biomedical materials. Due to the polyurethane structure features and foam technology it is possible to incorporate into their structure the biologically active compounds of target purpose via structural-chemical modification of macromolecule. A series of new biologically active polyurethane foams (PUFs) was synthesized with polyethers (MM 2500-5000), polyesters MM (500-2200), 2,4(2,6) toluene diisocyanate, water as a foaming agent, catalysts, foam stabilizers and functional compounds. Different functional compounds: 1,4-di-N-oxy-2,3-bis-(oxymethyl)-quinoxaline (DOMQ), partial sodium salt of poly(acrylic acid) and 2,6-dimethyl-N,N-diethyl aminoacetatanilide hydrochloride were incorporated into the polymer structure/composition due to the chemical and/or physical bonding. Structural peculiarities of PUFs were studied by FTIR spectroscopy and X-ray scattering. Self-adhesion properties of PUFs were estimated by measuring of tensile strength at break of adhesive junction. The optical microscopy method was performed for the PUF morphology studies. Toxicological estimation of the PUFs was carried out in vitro and in vivo. The antibacterial action towards the Gram-positive and Gram-negative bacteria (Escherichia coli ATC 25922, E. coli ATC 2150, Klebsiella pneumoniae 6447, Staphylococcus aureus 180, Pseudomonas aeruginosa 8180, Proteus mirabilis F 403, P. mirabilis 6054, and Proteus vulgaris 8718) was studied by the disc method on the solid nutrient. Physic-chemical properties of the PUFs (density, tensile strength and elongation at break, water absorption and vapor permeability) showed that all studied PUFs are within the operational requirements for such materials and represent fine-cellular foams. Spectral studies confirmed the incorporation of DOMQ into the PUF's macrochain. PUFs are characterized by microheterogeneous structure. They are antibacterially active, non

Multidimensional fractionation is a requirement for quantitation of Golgi-resident glycosylation enzymes from cultured human cells.

Science.gov (United States)

Lin, Chi-Hung; Chik, Jenny H L; Packer, Nicolle H; Molloy, Mark P

2015-02-06

Glycosylation results from the concerted action of glycosylation enzymes in the secretory pathway. In general, gene expression serves as the primary control mechanism, but post-translational fine-tuning of glycosylation enzyme functions is often necessary for efficient synthesis of specific glycan epitopes. While the field of glycomics has rapidly advanced, there lacks routine proteomic methods to measure expression of specific glycosylation enzymes needed to fill the gap between mRNA expression and the glycomic profile in a "reverse genomics" workflow. Toward developing this workflow we enriched Golgi membranes from two human colon cancer cell lines by sucrose density centrifugation and further mass-based fractionation by SDS-PAGE. We then applied mass spectrometry to demonstrate a doubling in the number of Golgi resident proteins identified, compared to the unenriched, low speed centrifuged supernatant of lysed cells. A total of 35 Golgi-resident glycosylation enzymes, of which 23 were glycosyltransferases, were identified making this the largest protein database so far of Golgi resident glycosylation enzymes experimentally identified in cultured human cells. We developed targeted mass spectrometry assays for specific quantitation of many of these glycosylation enzymes. Our results show that alterations in abundance of glycosylation enzymes at the protein level were generally consistent with the resultant glycomic profiles, but not necessarily with the corresponding glycosyltransferase mRNA expression as exemplified by the case of O-glycan core 1 T synthase.

Biological Activities of Hydrazone Derivatives

Directory of Open Access Journals (Sweden)

S. Güniz Küçükgüzel

2007-08-01

Full Text Available There has been considerable interest in the development of novel compounds with anticonvulsant, antidepressant, analgesic, antiinflammatory, antiplatelet, antimalarial, antimicrobial, antimycobacterial, antitumoral, vasodilator, antiviral and antischistosomiasis activities. Hydrazones possessing an azometine -NHN=CH- proton constitute an important class of compounds for new drug development. Therefore, many researchers have synthesized these compounds as target structures and evaluated their biological activities. These observations have been guiding for the development of new hydrazones that possess varied biological activities.

Biochemical studies on certain biologically active nitrogenous compounds

International Nuclear Information System (INIS)

Abdel kader, S.M.; El Sayed, M.M.; El Malt, E.A.; Shaker, E.S.; Abdel Aziz, H.G.

2010-01-01

Certain biologically active nitrogenous compounds such as alkaloids are widely distributed in many wild and medicinal plants such as peganum harmala L. (Phycophyllaceae). However, less literature cited on the natural compounds was extracted from the aerial parts of this plant; therefore this study was conducted on harmal leaves using several solvents. Data indicated that methanol extract was the inhibitoriest effect against some pathogenic bacteria, particularly Streptococcus pyogenus. Chromatographic separation illustrated that presence of four compounds; the most active one was the third compound (3). Elementary analysis (C, H, N) revealed that the primary chemical structure of the active antibacterial compound (C3) was: C17 H21 N3 O7 S with molecular weight 411. Spectroscopic analysis proved that coninical structure was = 1- thioformyl, 8?- D glucoperanoside- Bis- 2, 3 dihydroisopyridino pyrrol. This new compound is represented as a noval ?- carboline alkaloid compound

Marked increase in rat red blood cell membrane protein glycosylation by one-month treatment with a cafeteria diet

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Laia Oliva

2015-07-01

Full Text Available Background and Objectives. Glucose, an aldose, spontaneously reacts with protein amino acids yielding glycosylated proteins. The compounds may reorganize to produce advanced glycosylation products, which regulatory importance is increasingly being recognized. Protein glycosylation is produced without the direct intervention of enzymes and results in the loss of function. Glycosylated plasma albumin, and glycosylated haemoglobin are currently used as index of mean plasma glucose levels, since higher glucose availability results in higher glycosylation rates. In this study we intended to detect the early changes in blood protein glycosylation elicited by an obesogenic diet.Experimental Design. Since albumin is in constant direct contact with plasma glucose, as are the red blood cell (RBC membranes, we analyzed their degree or glycosylation in female and male rats, either fed a standard diet or subjected to a hyper-energetic self-selected cafeteria diet for 30 days. This model produces a small increase in basal glycaemia and a significant increase in body fat, leaving the animals in the initial stages of development of metabolic syndrome. We also measured the degree of glycosylation of hemoglobin, and the concentration of glucose in contact with this protein, that within the RBC. Glycosylation was measured by colorimetric estimation of the hydroxymethylfurfural liberated from glycosyl residues by incubation with oxalate.Results. Plasma glucose was higher in cafeteria diet and in male rats, both independent effects. However, there were no significant differences induced by sex or diet in either hemoglobin or plasma proteins. Purified RBC membranes showed a marked effect of diet: higher glycosylation in cafeteria rats, which was more marked in females (not in controls. In any case, the number of glycosyl residues per molecule were higher in hemoglobin than in plasma proteins (after correction for molecular weight. The detected levels of glucose in

Synthesis and Biological Activity of Novel Amino Acid-(N'-Benzoyl Hydrazide and Amino Acid-(N'-Nicotinoyl Hydrazide Derivatives

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Sherine N. Khattab

2005-09-01

Full Text Available The coupling reaction of benzoic acid and nicotinic acid hydrazides with N- protected L-amino acids including valine, leucine, phenylalanine, glutamic acid and tyrosine is reported. The target compounds, N-Boc-amino acid-(N`-benzoyl- and N- Boc-amino acid-(N`-nicotinoyl hydrazides 5a-5e and 6a-6e were prepared in very high yields and purity using N-[(dimethylamino-1H-1,2,3-triazolo[4,5-b]pyridin-1-yl- methylene]-N-methyl-methanaminium hexafluorophosphate N-oxide (HATU as coupling reagent. The antimicrobial activity of the Cu and Cd complexes of the designed compounds was tested. The products were deprotected affording the corresponding amino acid-(N`-benzoyl hydrazide hydrochloride salts (7a-7e and amino acid-(N`- nicotinoyl hydrazide hydrochloride salts (8a-8e. These compounds and their Cu and Cd complexes were also tested for their antimicrobial activity. Several compounds showed comparable activity to that of ampicillin against S. aureus and E. coli.

Patterns of glycemic control using glycosylated hemoglobin in diabetics

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Arunpreet Singh Kahlon

2011-01-01

Full Text Available Aim : Till now estimation of blood glucose is the highly effective method for diagnosing diabetes mellitus but it provides a short-term picture of control. More evidence is required to prove that plasma glucose and glycosylated hemoglobin levels together gives a better estimate of glycemic control and compliance with treatment. Indian diabetes risk score (IDRS is a simplified screening tool for identifying undiagnosed diabetic subjects, requires minimum time, and effort and can help to considerably reduce the costs of screening. Objective : To study patterns of glycemic control using glycosylated hemoglobin in diabetic patients. To find out correlation between levels of plasma glucose and glycosylated hemoglobin in diabetics and to calculate IDRS of the study population. Materials and Methods : A cross sectional study was conducted among 300 known diabetic patients attending outpatient department of a rural medical college in Haryana, India. Following standard procedures and protocols FPG and glycosylated hemoglobin were measured to find out a pattern of glycemic control in them after taking their written and informed consent. A correlation between the levels of glycosylated hemoglobin and fasting blood glucose was also calculated. These patients were made to fill a performa and their demographic and clinical risk factors were noted and based on this, their IDRS was calculated. This was done to validate the IDRS in Indian rural population. Results : Fifty-two percent of the population had fasting plasma glucose level between 125-150 mg/dl, 21% had this level between 151-175 mg/dl. Thirteen percent of the study subjects had HbA1C between 6.5-7.5, more than half (57.3% had this value between 7.5-8.5, 12% and 18% had values between 8.5-9.5 and 9.5-10.5, respectively. Twelve percent of the participants had HbA1C level higher than 10.5. Correlation of fasting plasma glucose level and HbA1C was also studied and found that correlation coefficient came

Glucansucrase Gtf180-ΔN of Lactobacillus reuteri 180 : enzyme and reaction engineering for improved glycosylation of non-carbohydrate molecules

NARCIS (Netherlands)

Devlamynck, Tim; Te Poele, Evelien M; Meng, Xiangfeng; van Leeuwen, Sander S; Dijkhuizen, Lubbert

2016-01-01

Glucansucrases have a broad acceptor substrate specificity and receive increased attention as biocatalysts for the glycosylation of small non-carbohydrate molecules using sucrose as donor substrate. However, the main glucansucrase-catalyzed reaction results in synthesis of α-glucan polysaccharides

Soil biological activity as affected by tillage intensity

Science.gov (United States)

Gajda, A. M.; Przewłoka, B.

2012-02-01

The effect of tillage intensity on changes of microbiological activity and content of particulate organic matter in soil under winter wheat duirng 3 years was studied. Microbial response related to the tillage-induced changes in soil determined on the content of biomass C and N, the rate of CO2 evolution, B/F ratio, the activity of dehydrogenases, acid and alkaline phosphatases, soil C/N ratio and microbial biomass C/N ratio confirmed the high sensitivity of soil microbial populations to the tillage system applied. After three year studies, the direct sowing system enhanced the increase of labile fraction of organic matter content in soil. There were no significant changes in the labile fraction quantity observed in soil under conventional tillage. Similar response related to the tillage intensity was observed in particulate organic matter quantities expressed as a percentage of total organic matter in soil. A high correlation coefficients calculated between contents of soil microbial biomass C and N, particulate organic matter and potentially mineralizable N, and the obtained yields of winter wheat grown on experimental fields indicated on a high importance of biological quality of status of soil for agricultural crop production.

NEW DERIVATIVES OF 2-R1-N-(5-R-1,3,4-THIADIAZOL-2-YL-BENZOLSULFONAMIDES: SYNTHESIS, PHYSICOCHEMICAL PROPERTIES AND BIOLOGICAL ACTIVITY PREDICTION

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Sych I.V.

2015-12-01

Full Text Available Introduction: The analysis of modern literature, including overseas one, showed that a lot of the scientific researches is devoted to finding and creating biologically active compounds on base 1,3,4-thiadiazole. Derivatives of 1,3,4-thiadiazole are the large group of heterocyclic compounds with high rates of antimicrobial, antituberculosis, antidiabetic, antineoplastic and anticonvulsant activity. Material and methods: The purpose of this study was the expansion of sulfone derivatives substituted nitrogen-containing heterocyclic systems through the synthesis of 2-R1-N (5-R-1,3,4-thiadiazol-2-ilbenzolsulfonamides and prediction their pharmacological activity for future planning pharmacological screening. Synthesis of semi-products 2-amino-5-R-1,3,4-thiadiazoles was carried out by cyclization thiosemicarbazide and substituted derivatives of carboxylic acids in the presence of concentrated sulfuric acid. The synthesis of target compounds 2-R1-N(5-R-1,3,4-thiadiazol-2-ylbenzolsulfon-amides was carried out by N-acylation of 2-amino-5R-1,3,4-thiadiazole substituted benzolsul-fochlorides in the presence of anhydrous pyridine. The reaction proceeds by the classic SN2-mechanism. The resulting compounds are white crystalline substances, soluble in alcohol, chloroform and acetone, difficult to dissolve in water. Yields of obtained compounds was satisfactory (76-84%. The purity of the obtained compounds was determined by TLC. The structure of the obtained compounds was proved by elemental analysis, IR methods and 1H NMR spectroscopy. NMR 1H spectra were recorded at Bruker WM spectrometer (200 MHz; solvent DMSO-d6; chemical shifts were in ppm, internal standard (TMS (tetramethylsilane was used. The prognosis of biological activity for obtained compounds were carried out using the program PASS (Prediction of Activity Spectra for Substances in order to plan the further pharmacological screening. The program PASS predicts more than 500 kinds of biological

Loss of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 3 and reduced O-glycosylation in colon carcinoma cells selected for hepatic metastasis

DEFF Research Database (Denmark)

Kato, Kentaro; Takeuchi, Hideyuki; Kanoh, Akira

2010-01-01

O-glycosylation of mucin is initiated by the attachment of N-acetyl-D-galactosamine (GalNAc) to serine or threonine residues in mucin core polypeptides by UDPGalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts). It is not well understood how GalNAc attachment is regulated by multiple...... ppGalNAc-Ts in each cell. In the present study, the expression levels of murine ppGalNAc-Ts (mGalNAc-Ts), T1, T2, T3, T4, T6, and T7 were compared between mouse colon carcinoma colon 38 cells and variant SL4 cells, selected for their metastatic potentials, by using the competitive RT-PCR method....... The expression levels of mGalNAc-T1, T2, and T7 were slightly higher in the SL4 cells than in the colon 38 cells, whereas the expression level of mGalNAc-T3 in the SL4 cells was 1.5% of that in the colon 38 cells. Products of enzymatic incorporations of GalNAc residues into FITCPTTTPITTTTK peptide by the use...

The 'sweet' spot of cellular pluripotency: protein glycosylation in human pluripotent stem cells and its applications in regenerative medicine.

Science.gov (United States)

Wang, Yu-Chieh; Lin, Victor; Loring, Jeanne F; Peterson, Suzanne E

2015-05-01

Human pluripotent stem cells (hPSCs) promise for the future of regenerative medicine. The structural and biochemical diversity associated with glycans makes them a unique type of macromolecule modification that is involved in the regulation of a vast array of biochemical events and cellular activities including pluripotency in hPSCs. The primary focus of this review article is to highlight recent advances in stem cell research from a glycobiological perspective. We also discuss how our understanding of glycans and glycosylation may help overcome barriers hindering the clinical application of hPSC-derived cells. A literature survey using NCBI-PubMed and Google Scholar was performed in 2014. Regenerative medicine hopes to provide novel strategies to combat human disease and tissue injury that currently lack effective therapies. Although progress in this field is accelerating, many critical issues remain to be addressed in order for cell-based therapy to become a practical and safe treatment option. Emerging evidence suggests that protein glycosylation may significantly influence the regulation of cellular pluripotency, and that the exploitation of protein glycosylation in hPSCs and their differentiated derivatives may lead to transformative and translational discoveries for regenerative medicine. In addition, hPSCs represent a novel research platform for investigating glycosylation-related disease.

Novel glycosylated mycosporine-like amino acid, 13-O-(β-galactosyl)-porphyra-334, from the edible cyanobacterium Nostoc sphaericum-protective activity on human keratinocytes from UV light.

Science.gov (United States)

Ishihara, Kenji; Watanabe, Ryuichi; Uchida, Hajime; Suzuki, Toshiyuki; Yamashita, Michiaki; Takenaka, Hiroyuki; Nazifi, Ehsan; Matsugo, Seiichi; Yamaba, Minami; Sakamoto, Toshio

2017-07-01

A UV-absorbing compound was purified and identified as a novel glycosylated mycosporine-like amino acid (MAA), 13-O-β-galactosyl-porphyra-334 (β-Gal-P334) from the edible cyanobacterium Nostoc sphaericum, known as "ge xian mi" in China and "cushuro" in Peru. Occurrence of the hexosylated derivative of shinorine (hexosyl-shinorine) was also supported by LC-MS/MS analysis. β-Gal-P334 accounted for about 86.5% of total MAA in N. sphaericum, followed by hexosyl-shinorine (13.2%) and porphyra-334 (0.2%). β-Gal-P334 had an absorption maximum at 334nm and molecular absorption coefficient was 46,700 at 334nm. Protection activity of β-Gal-P334 from UVB and UVA+8-methoxypsoralen induced cell damage on human keratinocytes (HaCaT) was assayed in comparison with other MAA (porphyra-334, shinorine, palythine and mycosporine-glycine). The UVB protection activity was highest in mycosporine-glycine, followed by palythine, β-Gal-P334, porphyra-334 and shinorine in order. β-Gal-P334 had highest protection activity from UVA+8-methoxypsoralen induced cell damage followed by porphyra-334, shinorine, mycosporine-glycine and palythine. We also found an antioxidant (radical-scavenging) activity of β-Gal-P334 by colorimetric and ESR methods. From these findings, β-Gal-P334 was suggested to play important roles in stress tolerant mechanisms such as UV and oxidative stress in N. sphaericum as a major MAA. We also consider that the newly identified MAA, β-Gal-P334 has a potential for use as an ingredient of cosmetics and toiletries. Copyright © 2017 Elsevier B.V. All rights reserved.

Glycosyl Cross-Coupling of Anomeric Nucleophiles: Scope, Mechanism, and Applications in the Synthesis of Aryl C-Glycosides.

Science.gov (United States)

Zhu, Feng; Rodriguez, Jacob; Yang, Tianyi; Kevlishvili, Ilia; Miller, Eric; Yi, Duk; O'Neill, Sloane; Rourke, Michael J; Liu, Peng; Walczak, Maciej A

2017-12-13

position. Taken together, facile access to both anomers of various glycoside nucleophiles, a broad reaction scope, and uniformly high transfer of anomeric configuration make the glycosyl cross-coupling reaction a practical tool for the synthesis of bioactive natural products, drug candidates, allowing for late-stage glycodiversification studies with small molecules and biologics.

Congenital disorders of glycosylation: The Saudi experience.

Science.gov (United States)

Alsubhi, Sarah; Alhashem, Amal; Faqeih, Eissa; Alfadhel, Majid; Alfaifi, Abdullah; Altuwaijri, Waleed; Alsahli, Saud; Aldhalaan, Hesham; Alkuraya, Fowzan S; Hundallah, Khalid; Mahmoud, Adel; Alasmari, Ali; Mutairi, Fuad Al; Abduraouf, Hanem; AlRasheed, Layan; Alshahwan, Saad; Tabarki, Brahim

2017-10-01

We retrospectively reviewed Saudi patients who had a congenital disorder of glycosylation (CDG). Twenty-seven Saudi patients (14 males, 13 females) from 13 unrelated families were identified. Based on molecular studies, the 27 CDG patients were classified into different subtypes: ALG9-CDG (8 patients, 29.5%), ALG3-CDG (7 patients, 26%), COG6-CDG (7 patients, 26%), MGAT2-CDG (3 patients, 11%), SLC35A2-CDG (1 patient), and PMM2-CDG (1 patient). All the patients had homozygous gene mutations. The combined carrier frequency of CDG for the encountered founder mutations in the Saudi population is 11.5 per 10,000, which translates to a minimum disease burden of 14 patients per 1,000,000. Our study provides comprehensive epidemiologic information and prevalence figures for each of these CDG in a large cohort of congenital disorder of glycosylation patients. © 2017 Wiley Periodicals, Inc.

In silico determination of intracellular glycosylation and phosphorylation sites in human selectins: Implications for biological function

DEFF Research Database (Denmark)

Ahmad, I.; Hoessli, D.C.; Gupta, Ramneek

2007-01-01

Post-translational modifications provide the proteins with the possibility to perform functions in addition to those determined by their primary sequence. However, analysis of multifunctional protein structures in the environment of cells and body fluids is made especially difficult by the presence...... both modifications are likely to occur can also be predicted (YinYang sites), to suggest further functional versatility. Structural modifications of hydroxyl groups of P-, E-, and L-selectins have been predicted and possible functions resulting from such modifications are proposed. Functional changes...... of the three selectins are based on the assumption that transitory and reversible protein modifications by phosphate and O-GlcNAc cause specific conformational changes and generate binding sites for other proteins. The computer-assisted prediction of glycosylation and phosphorylation sites in selectins should...

Biological activities of Lavandula angustifolia essential oil

OpenAIRE

Bílková, Zuzana

2013-01-01

Zuzana Bílková, Biological activities of Lavandula angustifolia essential oil, Thesis, Charles University in Prague, Faculty of Pharmacy in Hradec Králové, thesis author: PharmDr. Jan Martin, PhD., Hradec Králové, 2013, 72 pages. The thesis called "Biological activities of Lavandula angustifolia essential oil" is interested in biological activities of Lavandula angustifolia essential oil, specifically antifungal, antioxidant, anti-inflammatory, cytotoxicity, nematicidal and repellency activit...

Activities ofWiN Slovakia

International Nuclear Information System (INIS)

Melicherova, T.

2015-01-01

WiN Slovakia activities of the last two years are presented. We always try to join professional and social aspects of events to improve not only our knowledge but mutual relationship and cooperation. List of activities: – The technical visit in the contaminated areas in Belarus. – The excursion to the chemical and radiation laboratories of Slovak Army and the biological and radiation department of Veterinary University in Kosice. – The Open Days in the Slovak Hydrometeorological Institute in Bratislava. – The course on radiation protection in Casta-Papiernicka. All such activities are appreciated by our members and create good platform for next professional and personal relationship. (author)

Characterisation of a novel homodimeric N-acetyl-β-D-glucosaminidase from Streptococcus gordonii

International Nuclear Information System (INIS)

Harty, Derek W.S.; Chen Yingjian; Simpson, Christine L.; Berg, Tracey; Cook, Simon L.; Mayo, John A.; Hunter, Neil; Jacques, Nicholas A.

2004-01-01

An N-acetyl-β-D-glucosaminidase (GcnA) from Streptococcus gordonii FSS2 was cloned and sequenced. GcnA had a deduced molecular mass of 72,120 Da. The molecular weight after gel-filtration chromatography was 140,000 Da and by SDS-PAGE was 70,000 Da, indicating that the native protein was a homodimer. The deduced amino acid sequence had significant homology to a glycosyl hydrolase from Streptococcus pneumoniae and the conserved catalytic domain of the Family 20 glycosyl hydrolases. GcnA catalysed the hydrolysis of the synthetic substrates, 4-methylumbelliferyl (4MU)-N-acetyl-β-D-glucosaminide, 4MU-N-acetyl-β-D-galactosaminide, 4-MU-β-D-N,N ' -diacetylchitobioside, and 4-MU-β-D-N,N ' ,N''-chitotrioside as well as the respective chito-oligosaccharides. GcnA was optimally active at pH 6.6 and 42 deg. C. The K m for 4-MU-β-D-N,N ' ,N''-chitotrioside, 45 μM, was the lowest for all the substrates tested. Hg 2+ , Cu 2+ , Fe 2+ , and Zn 2+ completely inhibited while Co 2+ , Mn 2+ , and Ni 2+ partially inhibited activity. S. gordonii FSS2 and a GcnA negative mutant grew equally well on chito-oligosaccharides as substrates. The S. gordonii sequencing projects indicate two further N-acetyl-β-D-glucosaminidase activities

Genome-scale metabolic model of Pichia pastoris with native and humanized glycosylation of recombinant proteins

DEFF Research Database (Denmark)

Irani, Zahra Azimzadeh; Kerkhoven, Eduard J.; Shojaosadati, Seyed Abbas

2016-01-01

prediction of protein yield, demonstrates the effect of the different types of N-glycosylation of protein yield, and can be used to predict potential targets for strain improvement. The model represents a step towards a more complete description of protein production in P. pastoris, which is required...... for using these models to understand and optimize protein production processes....

N-acylsulfonamides: Synthetic routes and biological potential in medicinal chemistry.

Science.gov (United States)

Ammazzalorso, Alessandra; De Filippis, Barbara; Giampietro, Letizia; Amoroso, Rosa

2017-12-01

Sulfonamide is a common structural motif in naturally occurring and synthetic medicinal compounds. The rising interest in sulfonamides and N-acyl derivatives is attested by the large number of drugs and lead compounds identified in last years, explored in different fields of medicinal chemistry and showing biological activity. Many acylsulfonamide derivatives were designed and synthesized as isosteres of carboxylic acids, being the characteristics of these functional groups very close. Starting from chemical routes to N-acylsulfonamides, this review explores compounds of pharmaceutical interest, developed as enzymatic inhibitors or targeting receptors. © 2017 John Wiley & Sons A/S.

Conformationally superarmed S-ethyl glycosyl donors as effective building blocks for chemoselective oligosaccharide synthesis in one pot

DEFF Research Database (Denmark)

Bandara, Mithila D.; Yasomanee, Jagodige P.; Rath, Nigam P.

2017-01-01

A new series of superarmed glycosyl donors has been investigated. It was demonstrated that the S-ethyl leaving group allows for high reactivity, which is much higher than that of equally equipped S-phenyl glycosyl donors that were previously investigated by our groups. The superarmed S......-ethyl glycosyl donors equipped with a 2-O-benzoyl group gave complete β-stereoselectivity. Utility of the new glycosyl donors has been demonstrated in a one-pot one-addition oligosaccharide synthesis with all of the reaction components present from the beginning...

Molecular partners of hNOT/ALG3, the human counterpart of the Drosophila NOT and yeast ALG3 gene, suggest its involvement in distinct cellular processes relevant to congenital disorders of glycosylation, cancer, neurodegeneration and a variety of further pathologies.

Science.gov (United States)

Hacker, Benedikt; Schultheiß, Christoph; Döring, Michael; Kurzik-Dumke, Ursula

2018-06-01

This study provides first insights into the involvement of hNOT/ALG3, the human counterpart of the Drosophila Neighbour of TID and yeast ALG3 gene, in various putative molecular networks. HNOT/ALG3 encodes two translated transcripts encoding precursor proteins differing in their N-terminus and showing 33% identity with the yeast asparagine-linked glycosylation 3 (ALG3) protein. Experimental evidence for the functional homology of the proteins of fly and man in the N-glycosylation has still to be provided. In this study, using the yeast two-hybrid technique we identify 17 molecular partners of hNOT-1/ALG3-1. We disclose the building of hNOT/ALG3 homodimers and provide experimental evidence for its in vivo interaction with the functionally linked proteins OSBP, OSBPL9 and LRP1, the SYPL1 protein and the transcription factor CREB3. Regarding the latter, we show that the 55 kDa N-glycosylated hNOT-1/ALG3-1 molecule binds the N-glycosylated CREB3 precursor but does not interact with CREB3's proteolytic products specific to the endoplasmic reticulum and to the nucleus. The interaction between the two partners is a prerequisite for the proteolytic activation of CREB3. In case of the further binding partners, our data suggest that hNOT-1/ALG3-1 interacts with both OSBPs and with their direct targets LRP1 and VAMP/VAP-A. Moreover, our results show that various partners of hNOT-1/ALG3-1 interact with its diverse post translationally processed products destined to distinct cellular compartments. Generally, our data suggest the involvement of hNOT-1/ALG3-1 in various molecular contexts determining essential processes associated with distinct cellular machineries and related to various pathologies, such as cancer, viral infections, neuronal and immunological disorders and CDG.

Low Density Lipoprotein Receptor Class A Repeats Are O-Glycosylated in Linker Regions

DEFF Research Database (Denmark)

Pedersen, Nis Borbye; Wang, Shengjun; Narimatsu, Yoshiki

2014-01-01

, which in wild-type CHO cells is glycosylated with the typical sialylated core 1 structure. The glycosites in linker regions of LDLR class A repeats are conserved in LDLR from man to Xenopus and found in other homologous receptors. O-Glycosylation is controlled by a large family of polypeptide Gal...

Prediction, conservation analysis, and structural characterization of mammalian mucin-type O-glycosylation sites

DEFF Research Database (Denmark)

Julenius, Karin; Mølgaard, Anne; Gupta, Ramneek

2005-01-01

could be predicted from averaged properties together with the fact that glycosylation sites are not precisely conserved indicates that mucin-type glycosylation in most cases is a bulk property and not a very site-specific one. NetOGlyc 3.1 is made available at www.cbs.dtu.dk/services/netoglyc....

A novel cerebello-ocular syndrome with abnormal glycosylation due to abnormalities in dolichol metabolism.

NARCIS (Netherlands)

Morava, E.; Wevers, R.A.; Cantagrel, V.; Hoefsloot, L.H.; Al-Gazali, L.; Schoots, J.; Rooij, A. van; Huijben, K.; Ravenswaaij-Arts, C.M.A. van; Jongmans, M.C.J.; Sykut-Cegielska, J.; Hoffmann, G.F.; Bluemel, P.; Adamowicz, M.; Reeuwijk, J. van; Ng, B.G.; Bergman, J.E.; Bokhoven, J.H.L.M. van; Korner, C.; Babovic-Vuksanovic, D.; Willemsen, M.A.A.P.; Gleeson, J.G.; Lehle, L.; Brouwer, A.P.M. de; Lefeber, D.J.

2010-01-01

Cerebellar hypoplasia and slowly progressive ophthalmological symptoms are common features in patients with congenital disorders of glycosylation type I. In a group of patients with congenital disorders of glycosylation type I with unknown aetiology, we have previously described a distinct phenotype

Analysis of urinary PSA glycosylation is not indicative of high-risk prostate cancer.

Science.gov (United States)

Barrabés, Sílvia; Llop, Esther; Ferrer-Batallé, Montserrat; Ramírez, Manel; Aleixandre, Rosa N; Perry, Antoinette S; de Llorens, Rafael; Peracaula, Rosa

2017-07-01

The levels of core fucosylation and α2,3-linked sialic acid in serum Prostate Specific Antigen (PSA), using the lectins Pholiota squarrosa lectin (PhoSL) and Sambucus nigra agglutinin (SNA), can discriminate between Benign Prostatic Hyperplasia (BPH) and indolent prostate cancer (PCa) from aggressive PCa. In the present work we evaluated whether these glycosylation determinants could also be altered in urinary PSA obtained after digital rectal examination (DRE) and could also be useful for diagnosis determinations. For this purpose, α2,6-sialic acid and α1,6-fucose levels of urinary PSA from 53 patients, 18 biopsy-negative and 35 PCa patients of different aggressiveness degree, were analyzed by sandwich ELLA (Enzyme Linked Lectin Assay) using PhoSL and SNA. Changes in the levels of specific glycosylation determinants, that in serum PSA samples were indicative of PCa aggressiveness, were not found in PSA from DRE urine samples. Although urine is a simpler matrix for analyzing PSA glycosylation compared to serum, an immunopurification step was necessary to specifically detect the glycans on the PSA molecule. Those specific glycosylation determinants on urinary PSA were however not useful to improve PCa diagnosis. This could be probably due to the low proportion of PSA from the tumor in urine samples, which precludes the identification of aberrantly glycosylated PSA. Copyright © 2017 Elsevier B.V. All rights reserved.

NetOglyc: prediction of mucin type O-glycosylation sites based on sequence context and surface accessibility

DEFF Research Database (Denmark)

Hansen, Jan Erik; Lund, Ole; Tolstrup, Niels

1998-01-01

-glycosylated serine and threonine residues in independent test sets, thus proving more accurate than matrix statistics and vector projection methods. Predicition of O-glycosylation sites in the envelope glycoprotein gp120 from the primate lentiviruses HIV-1, HIV-2 and SIV are presented. The most conserved O...... structure and surface accessibility. The sequence context of glycosylated threonines was found to differ from that of serine, and the sites were found to cluster. Non-clustered sites had a sequence context different from that of clustered sites. charged residues were disfavoured at postition -1 and +3......-glycosylation signals in these evolutionary-related glycoproteins were found in their first hypervariable loop, V1. However, the strain variation for HIV-1 gp120 was significant. A computer server, available through WWW or E-mail, has been developed for prediction of mucin type O-glycosylation sites in proteins based...

Role of structure and glycosylation of adsorbed protein films in biolubrication.

Directory of Open Access Journals (Sweden)

Deepak H Veeregowda

Full Text Available Water forms the basis of lubrication in the human body, but is unable to provide sufficient lubrication without additives. The importance of biolubrication becomes evident upon aging and disease, particularly under conditions that affect secretion or composition of body fluids. Insufficient biolubrication, may impede proper speech, mastication and swallowing, underlie excessive friction and wear of articulating cartilage surfaces in hips and knees, cause vaginal dryness, and result in dry, irritated eyes. Currently, our understanding of biolubrication is insufficient to design effective therapeutics to restore biolubrication. Aim of this study was to establish the role of structure and glycosylation of adsorbed protein films in biolubrication, taking the oral cavity as a model and making use of its dynamics with daily perturbations due to different glandular secretions, speech, drinking and eating, and tooth brushing. Using different surface analytical techniques (a quartz crystal microbalance with dissipation monitoring, colloidal probe atomic force microscopy, contact angle measurements and X-ray photo-electron spectroscopy, we demonstrated that adsorbed salivary conditioning films in vitro are more lubricious when their hydrophilicity and degree of glycosylation increase, meanwhile decreasing their structural softness. High-molecular-weight, glycosylated proteins adsorbing in loops and trains, are described as necessary scaffolds impeding removal of water during loading of articulating surfaces. Comparing in vitro and in vivo water contact angles measured intra-orally, these findings were extrapolated to the in vivo situation. Accordingly, lubricating properties of teeth, as perceived in 20 volunteers comprising of equal numbers of male and female subjects, could be related with structural softness and glycosylation of adsorbed protein films on tooth surfaces. Summarizing, biolubrication is due to a combination of structure and glycosylation

Brassinosteroids: synthesis and biological activities

Czech Academy of Sciences Publication Activity Database

Oklešťková, Jana; Rárová, Lucie; Kvasnica, Miroslav; Strnad, Miroslav

2015-01-01

Roč. 14, č. 6 (2015), s. 1053-1072 ISSN 1568-7767 R&D Projects: GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : Brassinosteroids * Chemical synthesis * Plant biological activity Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.686, year: 2015

Los itinerarios profesionales en Biología: un ejemplo de formación académica orientada a la inserción profesional Professional pathways in Biology: an example of professionally-oriented teaching

Directory of Open Access Journals (Sweden)

José Aramburu

2006-12-01

Full Text Available Uno de los retos de la Universidad es formar los profesionales necesarios para la sociedad en que está inserta. Este ajuste no es fácil de obtener debido a la rigidez de los planes de estudios oficiales y a las cambiantes necesidades sociales. Sin embargo, el próximo Espacio Europeo de Educación Superior tiene este empeño como uno de sus principales objetivos. En el presente artículo se presenta la experiencia de los itinerarios profesionales de la licenciatura en Biología de la Universitat Pompeu Fabra durante dos cursos académicos. Situados en el quinto curso del plan de estudios, permiten a los estudiantes una formación específica en uno de los ámbitos profesionales en los que pueden insertarse laboralmente. La experiencia muestra que, tras un período de uno o dos años de su graduación, el 83% de los egresados se encuentran realizando un trabajo remunerado, en la mayoría de los casos relacionado con la biología, mientras que un 9% realizan otros estudios (no doctorado. Sólo el 3% de los graduados están buscando trabajo de forma activa. En conclusión, los itinerarios profesionales pueden constituir una estrategia educativa adecuada para permitir que los licenciados en Biología se inserten con éxito en ocupaciones laborales acordes con su formación.One of the main challenges facing universities is to train professionals who possess the skills that society requires. This is difficult to achieve due to the rigidity of official curricula and the constantly changing needs of society. However, it remains one of the main goals of the Bologna process. This paper describes the experience of the professional pathways which form part of the biology degree offered by the Universitat Pompeu Fabra, over a period of two academic years. Professional pathways are an essential part of the fifth year syllabus and provide students with specific training in one of the professional settings in which they may eventually work. Our experience